Humanized or affinity-matured anti ang-2 antibody and uses thereof

ABSTRACT

An anti-Ang2 antibody or an antigen-binding fragment thereof that specifically binds to an angiogenesis-inducing factor Angiopoietin-2 (Ang2) and complexes with a Tie2 receptor through Ang2, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of copending U.S. patent applicationSer. No. 14/721,591 filed on May 26, 2015, which in turn claims thebenefit of Korean Patent Application No. 10-2014-0063251 filed on May26, 2014 in the Korean Intellectual Property Office, the entiredisclosure of which is hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: One 60,035 byte ASCII (Text) file named “739192 ST25.TXTrevised” created May 9 2018.

BACKGROUND OF THE INVENTION 1. Field

An anti-Ang2 antibody or an antigen-binding fragment thereof thatspecifically binds to an angiogenesis-inducing factor Angiopoietin-2(Ang2) and complexes with a Tie2 receptor through Ang2, and methods ofusing the same, are provided.

2. Description of the Related Art

Angiogenesis refers to a mechanism through which a new blood vessel isformed from a pre-existing blood vessel, and has been known to play animportant role in, e.g., the formation of organs, promoting normalphysiological growth, promoting wound healing and the like. Abnormalangiogenesis has been known to play a crucial role in diseases such astumor growth and metastasis, age-related macular degeneration, diabeticretinopathy, psoriasis, rheumatoid arthritis, and chronic inflammation.

Angiogenesis has been known to play an important role in tumor growthand metastasis, and various intensive researches on angiogenesismechanism for developing a new cancer drug have been going on bydeveloped countries and multinational pharmaceutical companies. One ofthe proteins that have been the target of research is Angiopoietin whichhas been known to be involved in blood vessel development andangiogenesis after birth. Known members of the angiopoietin familyinclude Ang-1, 2, 3 and 4.

Angiogenesis related to Angiopoietin-2(Ang2) in a cancer tissue isbelieved to occur as follows. First, for angiogenesis in the cancertissue, cooption wherein cancer cells select pre-existing blood vesselsto form new blood vessels in a cancer tissue occurs. Thereafter, bloodvessel regression during which the functions of the pre-existing bloodvessels are destroyed by Ang2 pathway occurs. The regression of thepre-existing vessels causes hypoxic environment within the cancertissue, which is an environment where the formation of new blood vesselsis possible. Under such conditions, the expression of vascularendothelial cell growth factor (VEGF) is increased, and new bloodvessels are thus formed.

Thus, Ang2 is of increasing importance as a target for developing anangiogenesis inhibitor, and there is a need of developing an effectiveand strong Ang2 targeting substance.

BRIEF SUMMARY OF THE INVENTION

Provided is an anti-Ang2 antibody or an antigen-binding fragment thereofthat specifically binds to an angiogenesis-inducing factor Ang2(Angiopoietin-2) and forms a complex with a Tie2 receptor via Ang2 toinduce the activation of the Tie2 receptor. The anti-Ang2 antibody canbe humanized and/or affinity-matured.

In one embodiment, the anti-Ang2 antibody or an antigen-binding fragmentthereof may comprise or consist essentially of:

a heavy chain variable region comprising a polypeptide (CDR-H1)comprising SEQ ID NO: 1, a polypeptide (CDR-H2) comprising SEQ ID NO:20, and a polypeptide (CDR-H3) comprising SEQ ID NO: 3;

a light chain variable region comprising a polypeptide (CDR-L1)comprising SEQ ID NO: 21, a polypeptide (CDR-L2) comprising SEQ ID NO:22, and a polypeptide (CDR-L3) comprising SEQ ID NO: 23; or

a combination of the heavy chain variable region and the light chainvariable region,

with the proviso that the anti-Ang2 antibody or an antigen-bindingfragment thereof does not comprise all of a polypeptide (CDR-H1)comprising SEQ ID NO: 1, a polypeptide (CDR-H2) comprising SEQ ID NO: 2,a polypeptide (CDR-H3) comprising SEQ ID NO: 3, a polypeptide (CDR-L1)comprising SEQ ID NO: 4, a polypeptide (CDR-L2) comprising SEQ ID NO: 5,and a polypeptide (CDR-L3) comprising SEQ ID NO: 6.

Another embodiment provides a pharmaceutical composition including theanti-Ang2 antibody or an antigen-binding fragment thereof and a carrier.

Another embodiment provides a method for inhibiting angiogenesis,including administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need of inhibiting angiogenesis.

Another embodiment provides a method for decreasing vascularpermeability, including administering the anti-Ang2 antibody or anantigen-binding fragment thereof to a subject in need of decreasingvascular permeability.

Another embodiment provides a method for inducing normal blood vesselformation, including administering the anti-Ang2 antibody or anantigen-binding fragment thereof to a subject in need of inducing normalblood vessel formation.

Another embodiment provides a method for preventing and/or treating adisease associated with angiogenesis, an increase in vascularpermeability, and/or a decrease in normal blood vessel formation,including administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need of preventing and/or treating adisease associated with angiogenesis, an increase in vascularpermeability.

Another embodiment provides a method for inhibiting Ang2 and/oractivating Tie2 receptor, including administering the anti-Ang2 antibodyor an antigen-binding fragment thereof to a subject in need ofinhibiting Ang2 and/or activating Tie2 receptor.

Another embodiment provides a method for detecting Ang2 and/ordiagnosing a disease associated with overexpression of Ang2, using theanti-Ang2 antibody or an antigen-binding fragment thereof.

Another embodiment provides a complex including the anti-Ang2 antibodyor an antigen-binding fragment thereof and Ang2, wherein the anti-Ang2antibody or an antigen-binding fragment thereof binds to Ang2.

Another embodiment provides a pharmaceutical composition including thecomplex including the anti-Ang2 antibody or an antigen-binding fragmentthereof and Ang2 and a carrier.

Another embodiment provides a method for activating Tie2 receptor,including administering the complex including the anti-Ang2 antibody oran antigen-binding fragment and Ang2 to a subject in need of activatingTie2 receptor.

Another embodiment provides a complex including the anti-Ang2 antibodyor an antigen-binding fragment thereof, Ang2, and Tie2 receptor, whereinthe anti-Ang2 antibody or an antigen-binding fragment thereof is boundto Ang2, and Ang2 is bound to Tie2 receptor, to form a complex.

Another embodiment provides a hybridoma cell line that produces ananti-Ang2 antibody or antigen-binding fragment thereof.

Another embodiment provides a polynucleotide encoding an anti-Ang2antibody or antigen binding fragment thereof, optionally in a vector.

Another embodiment provides a method of preparing the anti-Ang2 antibodyor antigen-binding fragment thereof by expressing a polynucleotideencoding the antibody or antigen-binding fragment in a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the binding level between Tie2 receptor andAng2 according to the concentration of anti-Ang2 antibody 10D6 measuredby Ang2-Tie2 competition ELISA, indicating that the anti-Ang2 antibody10D6 does not inhibit the binding between Tie2 receptor and Ang2.

FIG. 2 is a series of graphs showing the phosphorylation level of Aktthat participated in downstream signaling of Tie2 receptor, by humanizedand affinity-matured anti-Ang2 antibodies as compared to a control.

FIG. 3 is a schematic view of a process of overlap extension PCR forobtaining scFv library genes of 10D6 mutants where a desired CDR ismutated in one embodiment.

FIG. 4 is a schematic view of a process of overlap extension PCR forobtaining scFv library genes of 10D6 mutants where a desired CDR ismutated in another embodiment.

FIG. 5 is a graph showing the phosphorylation level of Akt thatparticipated in downstream signaling of Tie2 receptor, by humanized andaffinity-matured anti-Ang2 antibodies as compared to a control.

FIG. 6 is a graph of the ELISA results showing the formation of acomplex by the binding of an anti-Ang2 antibody with Ang2 and Tie2.

FIG. 7 is a three-dimensional structure of the complex of Ang2 and Tie2,showing the positions of loops 1-4.

DETAILED DESCRIPTION OF THE INVENTION

It is found that an antibody which specifically binds to Ang2 but doesnot inhibit binding between Ang2 and a Tie2 receptor and forms a complex(antibody/Ang2/Tie2) together with Ang2 and the Tie2 receptor has acharacteristic of inducing the dimerization of the antibody. Throughthis, it can induce the activation of the Tie2 receptor and itsdownstream signaling by effectively clustering the Tie2 receptor in theantibody/Ang2/Tie2 complex. The anti-Ang2 antibody inhibits Ang2 and hasa dual function of Ang2 neutralization (inhibition of angiogenesis) andthe normalization of blood vessels; that is, the antibody binds to Ang2to induce the intracellular internalization and degradation thereof,thereby lowering the level of circulating Ang2, and at the same time, itinduces Tie2 downstream signaling by binding to a Tie2 receptor via(i.e., indirectly to) Ang2 to activate the Tie2 receptor, similarly toAng1, and induces the normalization of blood vessels. The normalizationof blood vessels refers to the process by which abnormally formed bloodvessels in cancer cells are converted into a structurally andfunctionally normal status; that is, the blood vessels in cancer cellsregain normal structure and have decreased vascular permeability, andthus recover their normal function performance abilities.

Provided is an antibody targeting an angiogenesis-inducing factor Ang2and particularly, an anti-Ang2 antibody or its humanized and/oraffinity-matured form, which not only inhibits the functions of Ang2 byspecifically binding to Ang2 thereby inhibiting angiogenesis anddecreasing density of blood vessels in tumor tissue but also induces theactivation of Tie2 by allowing anti-Ang2 antibody-bound Ang2 to bindTie2, thereby structurally and/or functionally normalizing the bloodvessels. The anti-Ang2 antibody or its humanized and/or affinity-maturedform may bind Ang2 in such a way that Ang2 may still bind with Tie2. Theanti-Ang2 antibody or its humanized and/or affinity-matured form may notdirectly bind to Tie2 receptor, but it can form a complex with Tie2 bybinding Ang2 which, in turn, binds Tie2 receptor. The anti-Ang2 antibodyor its humanized and/or affinity-matured form has the effects oftreating diseases by binding to a Tie2 receptor together with Ang2 toactivate the Tie2 receptor and thus induce the structural/functionalnormalization of blood vessels, along with the down-regulation of Ang2in diseases related to the dysfunction and the abnormal activation ofblood vessels such as cancer, sepsis, eye disorders, etc.

One embodiment provides an anti-Ang2 antibody or an antigen-bindingfragment thereof, specifically recognizing and/or binding to anangiogenesis-inducing factor Ang2 (Angiopoietin-2) and binding to a Tie2receptor via Ang2. Also, the anti-Ang2 antibody or an antigen-bindingfragment thereof may induce the activation of the Tie2 receptor. Suchactivation of Tie2 receptor may be induced by an increase in thephosphorylation of Tie2 receptor and/or the phosphorylation of proteinsrelated to the downstream signal pathway thereof, for example, at leastone selected from the group consisting of Akt (NM_005163), eNOS(NM_000603), 42/44 (NM_002745), etc. Also, the anti-Ang2 antibody or anantigen-binding fragment thereof may induce the intracellularinternalization of a Tie2 receptor. In other words, the anti-Ang2antibody or an antigen-binding fragment thereof may bind to Ang2 and theTie2 receptor via Ang2 to form a complex and induce the activation ofthe Tie2 receptor, by not inhibiting binding between Ang2 and the Tie2receptor while specifically binding to Ang2, unlike other types ofanti-Ang2 antibodies. Therefore, the anti-Ang2 antibody or anantigen-binding fragment thereof may increase the phosphorylation of aprotein related to the downstream signal pathway of Tie2 receptor, suchas at least one selected from the group consisting of Akt, eNOS, and42/44, compared to the case using other anti-Ang2 antibodies thatinhibit the binding between Ang2 and Tie2 receptor, such as antibody4H10 (SEQ ID NOs: 12 & 13), etc.

The anti-Ang2 antibody may be humanized and/or affinity-matured, therebyhaving more increased binding affinity to Ang2 and more effectivefunctions.

The Ang2 protein which functions as an antigen against the antibody isclosely related to angiogenesis, and as a soluble ligand present inblood, it is widely involved in angiogenesis, metastasis, cancer cellinvasion, etc. The Ang2 may be derived from mammals including primatessuch as humans and monkeys and rodents such as rats and mice and forexample, it may be human Ang2 (e.g., NCBI Accession No. O15123, etc.),monkey Ang2 (e.g., NCBI Accession No. Q8MIK6, etc.), mouse Ang2 (e.g.,NCBI Accession No. O35608, etc.), and rat Ang2 (e.g., NCBI Accession No.O35462, etc.), but is not limited thereto.

The Tie2 receptor (TEK tyrosine kinase), which is an Angiopoietin-1receptor, is expressed in vascular endothelial cells in various mammalssuch as mouse (NM_013690; NP_038718), rat, and human (NM_000459;NP_000450), and is involved in various downstream signaling.

As explained above, the anti-Ang2 antibody or an antigen-bindingfragment thereof is characterized in that the antibody whichspecifically binds to Ang2 but does not inhibit binding between Ang2 andTie2 receptor and forms a complex (antibody/Ang2/Tie2) together withAng2 and the Tie2 receptor, has a characteristic of inducing thedimerization of the antibody, and through this, it can induce theactivation of the Tie2 receptor and its downstream signaling byeffectively clustering the Tie2 receptor which constitutes the complex.By virtue of such an action mechanism, the anti-Ang2 antibody and theantigen-binding fragment thereof inhibits Ang2 functions by binding toAng2 to induce the intracellular internalization and degradation thereofand thus lowers the level of circulating Ang2 and at the same time, itinduces Tie2 downstream signaling by binding to the Tie2 receptortogether with Ang2 to activate the Tie2 receptor, like Ang1 and inducesthe stabilization of vascular endothelial cells. By having such dualfunctions, the antibody and the antigen-binding fragment thereof can beusefully employed to treat not only symptoms (disorders) due to theoverexpression of Ang2 but also symptoms (disorders) due to the decreasein the stabilization of vascular endothelial cells, that is, theincrease of vascular penetration.

The anti-Ang2 antibody or an antigen-binding fragment thereof mayrecognize all or part (for example, at least one amino acid selectedfrom the group consisting of the amino acid residues exposed to theoutside) of loop 1 (of SEQ ID NO: 11, a site from 417^(th) amino acid to434^(th) amino acid) of human Ang2 (hAng2; SEQ ID NO: 11; Accession#O15123) or an amino acid sequence site including about 2 to about 20,about 2 to about 15, 2 to about 10, or about 2 to about 5 contiguousamino acids including at least one exposed amino acid residue of loop 1of SEQ ID NO: 11 as an epitope. As used herein, the term “exposed” aminoacid is an amino acid that is exposed to solution (e.g., a biologicalmedium or other solution) and available for binding when a protein(e.g., Ang2) is in its native conformation in a biological medium orother solution under physiological conditions (e.g., physiological pH,isotonicity, temperature, etc.)

Ang2 (SEQ ID NO: 11) MWQIVFFTLS CDLVLAAAYN NFRKSMDSIG KKQYQVQHGSCSYTFLLPEM DNCRSSSSPY VSNAVQRDAP LEYDDSVQRL QVLENIMENN TQWLMKLENYIQDNMKKEMV EIQQNAVQNQ TAVMIEIGTN LLNQTAEQTR KLTDVEAQVL NQTTRLELQLLEHSLSTNKL EKQILDQTSE INKLQDKNSF LEKKVLAMED KHIIQLQSIK EEKDQLQVLVSKQNSIIEEL EKKIVTATVN NSVLQKQQHD LMETVNNLLT MMSTSNSAKD PTVAKEEQISFRDCAEVFKS GHTTNGIYTL TFPNSTEEIK AYCDMEAGGG GWTIIQRRED GSVDFQRTWKEYKVGFGNPS GEYWLGNEFV SQLTNQQRYV LKIHLKDWEG NEAYSLYEHF YLSSEELNYRIHLKGLTGTA GKISSISQPG NDFSTKDGDN DKCICKCSQM LTGGWWFDAC GPSNLNGMYYPQRQNTNKFN GIKWYYWKGS GYSLKATTMM IRPADF

For example, the anti-Ang2 antibody may recognize Q418, P419, acombination of Q418 and P419 positioned at loop 1 of SEQ ID NO: 11, oran amino acid sequence site including about 2 to about 20, about 2 toabout 15, about 2 to about 10, or about 2 to about 5 contiguous aminoacids including the amino acid residue of Q418, P419, or combination ofQ418 and P419 of SEQ ID NO: 11, as an epitope, or specifically bind tothis site. In one embodiment, the anti-Ang2 antibody may recognize theamino acid residues of Q418 and P419 of SEQ ID NO: 11 as an epitope, orspecifically bind to this portion.

Q418, P419, or an amino acid sequence (epitope) including them, to whichthe anti-Ang2 antibody specifically binds, are exposed amino acidresidues positioned in loop 1 of the three dimensional structure ofAng2, and they do not participate in binding between Ang2 and Tie2receptor.

In Q418, P419, or an amino acid sequence (epitope) including them, towhich the anti-Ang2 antibody specifically binds, the term “contiguousamino acids” may refer to not only amino acids which are consecutive onprimary structure, but also amino acids which are adjacent to oneanother on the secondary, or tertiary structure of a protein.

Therefore, the anti-Ang2 antibody or an antigen-binding fragment thereofmay be (1) an anti-Ang2 antibody or an antigen-binding fragment thatspecifically recognizes and/or binds to the above described site as anepitope, or (2) a humanized and/or affinity-matured anti-Ang2 antibodyor an antigen-binding fragment of the anti-Ang2 antibody or anantigen-binding fragment of (1).

In a particular embodiment, the anti-Ang2 antibody or an antigen-bindingfragment thereof may comprise or consist essentially of:

at least one heavy chain complementarity determining region (CDR)selected from the group consisting of a polypeptide (CDR-H1) includingthe amino acid sequence of SEQ ID NO: 1, a polypeptide (CDR-H2)including the amino acid sequence of SEQ ID NO: 2, and a polypeptide(CDR-H3) including the amino acid sequence of SEQ ID NO: 3, or a heavychain variable region including the at least one heavy chaincomplementarity determining region;

at least one light chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-L1) including the aminoacid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) including theamino acid sequence of SEQ ID NO: 5, and a polypeptide (CDR-L3)including the amino acid sequence of SEQ ID NO: 6, or a light chainvariable region including the at least one light chain complementaritydetermining region;

a combination of said at least one heavy chain complementaritydetermining region and said at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

More particularly, the anti-Ang2 antibody or an antigen-binding fragmentthereof may comprise or consist essentially of:

a heavy chain variable region including a polypeptide (CDR-H1) includingthe amino acid sequence of SEQ ID NO: 1, a polypeptide (CDR-H2)including the amino acid sequence of SEQ ID NO: 2, and a polypeptide(CDR-H3) including the amino acid sequence of SEQ ID NO: 3;

a light chain variable region including a polypeptide (CDR-L1) includingthe amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2)including the amino acid sequence of SEQ ID NO: 5, and a polypeptide(CDR-L3) including the amino acid sequence of SEQ ID NO: 6;

a combination of the heavy chain variable region and the light chainvariable region.

In one embodiment, the heavy chain variable region of the antibody orthe antigen-binding fragment thereof may include the amino acid sequenceof SEQ ID NO: 7 or 84:

(SEQ ID NO: 7) DVQLQESGPDLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMGYINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTATYYCAR GN FEGAMDYWGQGTSVTVSS; (SEQ ID NO: 84) DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWNWIRQFPGNKLEWMG YINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTATYYCAR GNFEGAMDY WGQGTLVTVSS

(In SEQ ID NO: 7 or 84 above, the underlined bold letters are CDRH1,CDRH2, and CDRH3 in sequence)

The light chain of the antibody according to one embodiment may includethe amino acid sequence of SEQ ID NO: 9:

(SEQ ID NO: 9) SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA WYQQKPGQSPKLLIYYASNRYP GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC QQDYSSPWT FGGGTKLEIK

(In SEQ ID NO: 9 above, the underlined bold letters are CDRL1, CDRL2,and CDRL3 in sequence)

The anti-Ang2 antibody or an antigen-binding fragment thereof mayinclude a heavy chain variable region including the amino acid sequenceof SEQ ID NO: 7 or 84, a light chain variable region including the aminoacid sequence of SEQ ID NO: 9, or a combination of the heavy chainvariable region and the light chain variable region.

For example, the anti-Ang2 antibody or an antigen-binding fragmentthereof may include a heavy chain variable region including the aminoacid sequence of SEQ ID NO: 7 or 84 and a light chain variable regionincluding the amino acid sequence of SEQ ID NO: 9.

The anti-Ang2 antibody or an antigen-binding fragment thereof may beaffinity-matured by substituting at least one amino acid residue of atleast one CDR, for example, at least one selected from the groupconsisting of CDR-H2, CDR-L1, CDR-L2 and CDR-L3, with other amino acids(i.e., with an amino acid that is different from the original one, whilemaintaining the inherent activity of the anti-Ang2 antibody).

For example, the affinity maturation of an anti-Ang2 antibody or anantigen-binding fragment may include at least one of the followingsubstitutions:

(1) a substitution of the 1^(st) amino acid residue Tyr (Y) of the aminoacid sequence of SEQ ID NO: 2 (YINYSGNTDYNPSLKS) of CDR-H2, with Lys(K);

(2) a substitution of the 3^(rd) amino acid residue Asn (N) of the aminoacid sequence of SEQ ID NO: 2 (YINYSGNTDYNPSLKS) of CDR-H2, with Ser(S);

(3) a substitution of the 5^(th) amino acid residue Ser (S) of the aminoacid sequence of SEQ ID NO: 2 (YINYSGNTDYNPSLKS) of CDR-H2, with Ala(A);

(4) a substitution of the 7^(th) amino acid residue Asn (N) of the aminoacid sequence of SEQ ID NO: 2 (YINYSGNTDYNPSLKS) of CDR-H2, with Lys(K);

(5) a substitution of the 11^(th) amino acid residue Ala (A) of theamino acid sequence of SEQ ID NO: 4 (KASQSVSNDVA) of CDR-L1, with His(H);

(6) a substitution of the 5^(th) amino acid residue Ser (S) of the aminoacid sequence of SEQ ID NO: 4 (KASQSVSNDVA) of CDR-L1, with Phe (F);

(7) a substitution of the 8^(th) amino acid residue Asn (N) of the aminoacid sequence of SEQ ID NO: 4 (KASQSVSNDVA) of CDR-L1, with Thr (T);

(8) a substitution of the 4^(th) amino acid residue Asn (N) of the aminoacid sequence of SEQ ID NO: 5 (YASNRYP) of CDR-L2, with Ile (I);

(9) a substitution of the 5^(th) amino acid residue Arg (R) of the aminoacid sequence of SEQ ID NO: 5 (YASNRYP) of CDR-L2, with Pro (P);

(10) a substitution of the 2^(nd) amino acid residue Gln (Q) of theamino acid sequence of SEQ ID NO: 6 (QQDYSSPWT) of CDR-L3, with His (H);and

(11) a substitution of the 8^(th) amino acid residue Trp (W) of theamino acid sequence of SEQ ID NO: 6 (QQDYSSPWT) of CDR-L3, with Phe (F),or

any combination thereof.

In an embodiment, the affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may comprise or consist essentially ofa polypeptide comprising an amino acid sequence of represented byfollowing general formula 1 (SEQ ID NO: 20) as a CDR-H2:

(General Formula 1) (SEQ ID NO: 20) X1-I-X2-Y-X3-G-X4-T-D-Y-N-P-S-L-K-S

wherein, X1 is Tyr (Y) or Lys (K), X2 is Asn (N) or Ser (S), X3 is Ser(S) or Ala (A), and X4 is Asn (N) or Lys (K).

For example, the amino acid sequence of SEQ ID NO: 20 may be provided bythe amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

In another embodiment, the affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may comprise or consist essentially ofa polypeptide comprising an amino acid sequence of represented byfollowing general formula 2 (SEQ ID NO: 21) as a CDR-L1:

(General Formula 2) (SEQ ID NO: 21) K-A-S-Q-X5-V-S-X6-D-V-X7

wherein, X5 is Ser (S) or Phe (F), X6 is Asn (N) or Thr (T), and X7 isAla (A) or His (H).

For example, the amino acid sequence of SEQ ID NO: 21 may be provided bythe amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17.

In another embodiment, the affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may comprise or consist essentially ofa polypeptide comprising an amino acid sequence of represented byfollowing general formula 3 (SEQ ID NO: 22) as a CDR-L2:

(General Formula 3) (SEQ ID NO: 22) Y-A-S-X8-X9-Y-P

wherein, X8 is Asn (N) or Ile (I) and X9 is Arg (R) or Pro (P).

For example, the amino acid sequence of SEQ ID NO: 22 may be provide bythe amino acid sequence of SEQ ID NO: 18.

In another embodiment, the affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may comprise or consist essentially ofa polypeptide comprising an amino acid sequence of represented byfollowing general formula 4 (SEQ ID NO: 23) as a CDR-L3:

(General Formula 4) (SEQ ID NO: 23) Q-X10-D-Y-S-S-P-X11-T

wherein, X10 is Gln (Q) or His (H) and X11 is Trp (W) or Phe (F).

For example, the amino acid sequence of SEQ ID NO: 23 may be provided bythe amino acid sequence of SEQ ID NO: 19.

In an embodiment, the affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may comprise or consist essentially of:

a heavy chain variable region including a polypeptide (CDR-H1) includingthe amino acid sequence of SEQ ID NO: 1, a polypeptide (CDR-H2)including the amino acid sequence of SEQ ID NO: 20, and a polypeptide(CDR-H3) including the amino acid sequence of SEQ ID NO: 3;

a light chain variable region including a polypeptide (CDR-L1) includingthe amino acid sequence of SEQ ID NO: 21, a polypeptide (CDR-L2)including the amino acid sequence of SEQ ID NO: 22, and a polypeptide(CDR-L3) including the amino acid sequence of SEQ ID NO: 23;

or

a combination of the heavy chain variable region and the light chainvariable region.

For example, the affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may comprise or consist essentially of:

a heavy chain variable region including a polypeptide (CDR-H1) includingthe amino acid sequence of SEQ ID NO: 1, a polypeptide (CDR-H2)including an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 14, and 15, and a polypeptide (CDR-H3) including theamino acid sequence of SEQ ID NO: 3;

a light chain variable region including a polypeptide (CDR-L1) includingan amino acid sequence selected from the group consisting of SEQ ID NOs:4, 16, and 17, a polypeptide (CDR-L2) including the amino acid sequenceof SEQ ID NO: 5 or 18, and a polypeptide (CDR-L3) including the aminoacid sequence of SEQ ID NO: 6 or 19;

or

a combination of the heavy chain variable region and the light chainvariable region.

In one embodiment, the affinity matured and/or humanized anti-Ang2antibody or an antigen-binding fragment thereof of the presentdescription does not include all of a polypeptide (CDR-H1) comprisingthe amino acid sequence of SEQ ID NO: 1, a polypeptide (CDR-H2)comprising the amino acid sequence of SEQ ID NO: 2, a polypeptide(CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, apolypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 4,a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO:5, and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQID NO: 6 together.

The complementarity determining regions of the anti-Ang2 antibody thatmay serve as a parent antibody or an antigen-binding fragment thereofand the affinity-matured anti-Ang2 antibody or an antigen-bindingfragment thereof are summarized in Table 1, as follows:

TABLE 1 Amino acid sequence of heavy chain CDR CDRH1-KABAT CDRH2-KABATCDRH3-KABAT Parent antibody SDYAWN (SEQ ID NO: 1) YINYSGNTDYNPSLKS (SEQGNFEGAMDY (SEQ ID ID NO: 2) NO: 3) Affinity-maturedKISYSGKTDYNPSLKS (SEQ antibody ID NO: 14) KINYAGNTDYNPSLKS (SEQID NO: 15) Amino acid sequence of light chain CDR CDRL1-KABATCDRL2-KABAT CDRL3-KABAT Parent antibody KASQSVSNDVA YASNRYP QQDYSSPWT(SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6) Affinity-maturedKASQSVSNDVH (SEQ YASIPYP (SEQ ID NO: 18) QHDYSSPFT (SEQ ID antibodyID NO: 16) NO: 19) KASQFVSTDVH (SEQ ID NO: 17)

For example, the affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may be selected from the groupconsisting of:

(a) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 14, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 4, CDR-L2 ofSEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6;

(b) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 14, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 16, CDR-L2of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6;

(c) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 15, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 4, CDR-L2 ofSEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6;

(d) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 15, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 16, CDR-L2of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6;

(e) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 2, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 16, CDR-L2of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 19;

(f) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 14, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 17, CDR-L2of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6;

(g) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 14, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 4, CDR-L2 ofSEQ ID NO: 18, and CDR-L3 of SEQ ID NO: 6;

(h) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain complementarity determining region comprising aCDR-H1 of SEQ ID NO: 1, a CDR-H2 of SEQ ID NO: 14, and a CDR-H3 of SEQID NO: 3, or a heavy chain variable region comprising the heavy chaincomplementarity determining region; and a light chain complementaritydetermining region comprising CDR-L1 of SEQ ID NO: 16, CDR-L2 of SEQ IDNO: 18, and CDR-L3 of SEQ ID NO: 6, or a light chain variable regioncomprising the light chain complementarity determining region; and

(i) an anti-Ang2 antibody or an antigen-binding fragment thereofcomprising a heavy chain variable region comprising a CDR-H1 of SEQ IDNO: 1, a CDR-H2 of SEQ ID NO: 14, and a CDR-H3 of SEQ ID NO: 3; and alight chain variable region comprising CDR-L1 of SEQ ID NO: 17, CDR-L2of SEQ ID NO: 18, and CDR-L3 of SEQ ID NO: 6n.

The affinity-matured anti-Ang2 antibody or an antigen-binding fragmentmay have an binding affinity (KD) to Ang2 of about 10 nM or less, about5 nM or less, about 2 nM or less, or about 1 nM or less, for example,about 0.01 to about 10 nM, about 0.01 to about 5 nM, about 0.01 to about2 nM, or about 0.01 to about 1 nM. The affinity-matured anti-Ang2antibody or an antigen-binding fragment shows a considerable improvementin the binding affinity (KD) to Ang2, because the binding affinity (KD)to Ang2 of its parent antibody anti-Ang2 antibody is about 8 nM.

In another embodiment, a humanized anti-Ang2 antibody or anantigen-binding fragment thereof is provided. The humanized anti-Ang2antibody or an antigen-binding fragment thereof may be obtained bysubstituting at least one amino acid residue of framework region (i.e.,the region other than the heavy chain complementarity determiningregion) of a heavy chain variable region (e.g., SEQ ID NO: 7 or 84). Theamino acid sequences of the framework region of a heavy chain variableregion, which can be used in producing a humanized anti-Ang2 antibody oran antigen-binding fragment thereof, are summarized in Table 2:

TABLE 2 (Humanization of a heavy chain) FR2 (framework FR3 (frameworkFR1 (framework region between region between FR4 (frameworkregion of N-terminus CDR-H1 and CDR- CDR-H2 and CDR-region of C-terminus of CDR-H1) H2) H3) of CDR-H3) Parent antibodyDVQLQESGPDLVK WIRQFPGNKLEW RSSITRDTSKNQFF WGQGTSVTVSS (SEQ (SEQ ID NO: 7PSQSLSLTCTVTG MG (SEQ ID NO: 29) LQLNSVTTGDTAT ID NO: 39) or or 84)YSIT (SEQ ID NO: YYCAR (SEQ ID WGQGTLVTVSS (SEQ 24) or NO: 34)ID NO: 86) DVQLQESGPGLVK PSQSLSLTCTVTG YSIT (SEQ ID NO: 85) HumanizedQVQLQESGPGLVK WIRQPPGKGLEWI RVTISVDTSKNQF WGQGTLVTVSS (SEQ antibody (VH-PSETLSLTCAVSG G (SEQ ID NO: 30) SLKLSSVTAADTA ID NO: 40) hu1)YSIS (SEQ ID NO: VYYCAR (SEQ ID 25) NO: 35) Humanized QVQLQESGPGLVKWIRQPPGKGLEW RSTISRDTSKNQFS WGQGTLVTVSS (SEQ antibody (VH- PSETLSLTCAVSGMG (SEQ ID NO: 31) LKLSSVTAADTAV ID NO: 41) hu2) YSIT (SEQ ID NO:YYCAR (SEQ ID 26) NO: 36) Humanized QVQLQESGPGLVK WIRQPPGKGLEWIRVTISVDTSKNQF WGQGTLVTVSS (SEQ antibody (VH- PSETLSLTCAVSGG(SEQ ID NO: 32) SLKLSSVTAADTA ID NO: 42) hu5) YSIT (SEQ ID NO:VYYCAR (SEQ ID 27) NO: 37) Humanized EVQLVESGGGLV WVRQAPGKGLEWRSTISRDTSKNTFY WGQGTLVTVSS (SEQ antibody (VH- QPGGSLRLSCAASMG (SEQ ID NO: 33) LQMNSLRAEDTA ID NO: 43) hu3) GYSIT (SEQ ID NO:VYYCAR (SEQ ID 28) NO: 38)

In addition, the humanized anti-Ang2 antibody or an antigen-bindingfragment thereof may be obtained by substituting at least one amino acidresidue of framework region (i.e., the region other than the heavy chaincomplementarity determining region) of a light chain variable region(e.g., SEQ ID NO: 9). The amino acid sequences of the framework regionof a light chain variable region, which can be used in producing ahumanized anti-Ang2 antibody or an antigen-binding fragment thereof, aresummarized in Table 3:

TABLE 3 (Humanization of a light chain) FR1 (framework FR2 (frameworkFR3 (framework FR4 (framework region adjacent to N- region between CDR-region between CDR- region adjacent to C- terminus of CDR-L1)L1 and CDR-L2) L2 and CDR-L3) terminus of CDR-L3) Parent SIVMTQTPKFLLVSWYQQKPGQSPKLL GVPDRFTGSGYGT FGGGTKLEIK (SEQ antibody (SEQ AGDRVTITC (SEQIY (SEQ ID NO: 46) DFTFTISTVQAEDL ID NO: 50) ID NO: 9) ID NO: 44)AVYFC (SEQ ID NO: 48) Humanized DIQMTQSPSSLSAS WYQQKPGKAPKLLGVPSRFSGSGSGTD FGQGTKVEIK (SEQ antibody (VL- VGDRVTITC (SEQIY (SEQ ID NO: 47) FTLTISSLQPEDFAT ID NO: 51) hu1) ID NO: 45)YYC (SEQ ID NO: 49)

In an embodiment, a heavy chain variable region of the humanizedanti-Ang2 antibody or an antigen-binding fragment thereof may compriseor consist essentially of:

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 24 to 28, for example, an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25 to 28, as aframework region adjacent to N-terminus of CDR-H1,

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 29 to 33, for example, an amino acid sequenceselected from the group consisting of SEQ ID NOs: 30 to 33, as aframework region between CDR-H1 and CDR-H2,

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 34 to 38, for example, an amino acid sequenceselected from the group consisting of SEQ ID NOs: 35 to 38, as aframework region between CDR-H2 and CDR-H3, and

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 39 to 43, for example, an amino acid sequenceselected from the group consisting of SEQ ID NOs: 40 to 43, as aframework region adjacent to C-terminus of CDR-H3.

A light chain variable region of the humanized anti-Ang2 antibody or anantigen-binding fragment thereof may comprise or consist essentially of:

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 44 or 45, for example, SEQ ID NO: 45, as aframework region adjacent to N-terminus of CDR-L1,

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 46 or 47, for example, SEQ ID NO: 47, as aframework region between CDR-L1 and CDR-L2,

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 48 or 49, for example, SEQ ID NO: 49, as aframework region between CDR-L2 and CDR-L3, and

a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 50 or 51, for example, SEQ ID NO: 51, as aframework region adjacent to C-terminus of CDR-L3.

In certain embodiments, one or more of the heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 24 as a frameworkregion adjacent to N-terminus of CDR-H1, the amino acid sequence of SEQID NO: 29 as a framework region between CDR-H1 and CDR-H2, the aminoacid sequence of SEQ ID NO: 34 as a framework region between CDR-H2 andCDR-H3, and the amino acid sequence of SEQ ID NO: 39 as a frameworkregion adjacent to C-terminus of CDR-H3; and the light chain variableregion comprising the amino acid sequence of SEQ ID NO: 44 as aframework region adjacent to N-terminus of CDR-L1, the amino acidsequence of SEQ ID NO: 46 as a framework region between CDR-L1 andCDR-L2, the amino acid sequence of SEQ ID NO: 48 as a framework regionbetween CDR-L2 and CDR-L3, and the amino acid sequence of SEQ ID NO: 50as a framework region adjacent to C-terminus of CDR-L3, can be excludedfrom the humanized anti-Ang2 antibody or an antigen-binding fragmentthereof according to the present description.

In an embodiment, the humanized anti-Ang2 antibody or an antigen-bindingfragment thereof may comprises a heavy chain variable region comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:52 to 56, a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 57 to 63, orany combination thereof.

The antibody includes any animal-derived antibodies, chimericantibodies, humanized antibodies and human antibodies. An animal-derivedantibody which is produced by immunizing an animal with a desiredantigen may generally trigger an immune rejection response whenadministered to humans for treatment purpose, and a chimeric antibodyhas been developed to suppress such immune rejection response. Achimeric antibody is formed by replacing the constant region of ananimal-derived antibody, which is a cause of anti-isotype response, withthe constant region of a human antibody using genetic engineeringmethods. The chimeric antibody has considerably improved anti-isotyperesponse in comparison with animal-derived antibodies, butanimal-derived amino acids are still present in its variable regions andthus it still contains potential side effects resulting from ananti-idiotypic response. It is a humanized antibody that has been thusdeveloped to improve such side effects. This is manufactured by graftingCDR (complementarity determining regions) which, of the variable regionsof a chimeric antibody, has an important role in antigen binding into ahuman antibody framework.

An important consideration in CDR grafting technology for manufacturinga humanized antibody is to select an optimized human antibody which canreceive best the CDR of an animal-derived antibody and for this,utilization of antibody database, analysis of crystal structure,molecule modeling technology, etc. are employed. However, although theCDR of an animal-derived antibody is grafted into an optimized humanantibody framework, there are a considerable number of cases whereantigen binding affinity is not preserved because there are amino acidswhich affect antigen binding while being positioned at the framework ofthe animal-derived antibody. In this regard, it may be essential toapply an additional antibody engineering technology for restoringantigen binding affinity.

According to one embodiment, the antibody may be an animal antibody suchas a mouse-derived antibody, a chimeric antibody such as a mouse-humanchimeric antibody, a humanized antibody, or a human antibody. Theantibody or an antigen-binding fragment thereof may be isolated from aliving body or non-naturally occurring. The antibody or anantigen-binding fragment thereof may be recombinant or synthetic. Theantibody or an antigen-binding fragment thereof may be monoclonal.

Antibodies have been widely used for treating diseases. As antibodiesare very stable in vivo as well as in vitro and have a long half-life,they are favorable for mass expression and production. Additionally,since an antibody has intrinsically a dimer structure, it has a fairlyhigh avidity.

An intact antibody has a structure with two full-length light chains andtwo full-length heavy chains, and each light chain is linked to eachheavy chain via a disulfide bond. The constant region of an antibody isdivided into a heavy chain constant region and a light chain constantregion, and the heavy chain constant region has gamma (γ), mu (μ), alpha(α), delta (δ) and epsilon (ε) types, and has gamma1 (γ1), gamma2 (γ2),gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2) as its subclass.The light chain constant region has kappa (κ) and lambda (λ) types.

The term “heavy chain” is understood to include a full-length heavychain and fragments thereof, the full-length heavy chain including avariable region domain V_(H) including an amino acid sequence havingsufficient variable region sequences that contribute the specificity forantigen binding and three constant region domains C_(H1), C_(H2) andC_(H3) domains and a hinge. The term “light chain” is understood toinclude a full-length light chain and fragments thereof, the full-lengthlight chain including a variable region domain V_(L) including an aminoacid sequence having sufficient variable region sequences thatcontribute to the specificity for antigen binding and a constant regiondomain C_(L).

The term “CDR (complementarity determining region)” refers to an aminoacid sequence found in the hypervariable region of a heavy chain and alight chain of an immunoglobulin. The heavy and light chain may eachinclude three CDRs (CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, CDRL3). TheCDRs of an antibody can provide an essential contact residue for bindingto an antigen or an epitope. Throughout the specification, the terms“specifically binding” or “specifically recognizing” have the samemeaning as generally known to an ordinary person in the art, indicatingthat an antigen and an antibody specifically interact with each other tolead to an immunological response.

The antigen-binding site of an antibody may be a fragment including atleast one complementarity determining region.

The term “antigen-binding fragment,” which is a fragment of the fullstructure of an immunoglobulin, refers to some of a polypeptideincluding a portion to which an antigen can bind. For example, it may bean scFv, an (scFv)₂, an scFv-Fc, an Fab, an Fab′ or an F(ab′)₂, but isnot limited thereto.

Among the above antigen-binding fragments, an Fab, which is a structurehaving the light chain and heavy chain variable regions, the light chainconstant region, and the heavy chain first constant region (C_(H1)), hasone antigen binding site. An Fab′ differs from the Fab in that the Fab′has a hinge region including at least one cysteine residue at theC-terminal of the heavy chain C_(H1) domain. An F(ab′)₂ is produced whencysteine residues at the hinge region of Fab′ are joined by a disulfidebond. An Fv is a minimal antibody fragment, having only heavy chainvariable regions and light chain variable regions, and a recombinanttechnique for producing the Fv fragment is well known in the art. In atwo-chain Fv fragment, the heavy chain variable domains are associatedwith the light chain variable domains via a non-covalent bond. Asingle-chain Fv fragment has a structure in which a heavy chain variabledomain and a light chain variable domain are covalently joined to eachother via a covalent bond or directly at the C-terminus, so that it canform a dimer as in a two-chain Fv fragment. In this context, the heavychain variable region and the light chain variable region may beconnected with each other through a linker, e. g., a peptide linker, ordirectly. The peptide linker may be composed of about 1 to about 100amino acid residues, or about 2 to about 50 amino acid residues. Forexample, the peptide linker may include Gly, Asn and/or Ser, and mayalso include neutral amino acids such as Thr and/or Ala. Amino acidsequences suitable for use in the peptide linker may be those well knownin the art. So long as it has no negative influence on the function ofthe antigen-binding fragment, the length of the peptide linker may beappropriately adjusted. For example, the peptide linker may be an aminosequence composed of about 1 to about 100, about 2 to about 50, or about5 to about 25 amino acid residues selected from among Gly, Asn, Ser,Thr, Ala, and a combination thereof. By way of example, the peptidelinker may be (GGGGS)n (wherein n represents the repeating number of(GGGGS) and may be an integer of 1 to 10, e.g., 2 to 5).

The antigen-binding fragment may be obtained using a protease (forexample, a whole antibody can be digested with papain to obtain Fabfragments, or can be digested with pepsin to obtain F(ab′)₂ fragments),or may be prepared by a genetic recombinant technique.

The term “hinge region” refers to a region included in the heavy chainsof an antibody, which is present between the CH1 and CH2 regions, andprovides flexibility to the antigen binding site in the antibody. Forexample, the hinge may be derived from a human antibody andparticularly, it may be derived from IgA, IgE, IgD, IgM, or IgG, forexample, IgG1, IgG2, IgG 3, or IgG4.

When an animal-derived antibody goes through a chimerization process, ananimal-derived IgG1 hinge is replaced with a human IgG1 hinge, but alength of the animal-derived IgG1 hinge is shorter than the human IgG1hinge, and disulfide bonds between two heavy chains are reduced from 3to 2. Thus, rigidity of the hinges may have different effects.Therefore, modification of a hinge region can increase an antigenbinding efficiency of a humanized antibody. Methods of deleting,inserting, or substituting an amino acid for modifying amino acidsequences of the hinge region are well known in the art.

Portions (e.g., constant regions) except the CDRs or variable regions ofthe anti-Ang2 antibody may be derived from an immunoglobulin such asIgA, IgD, IgE, IgD, IgM, or IgG, for example, IgG1, IgG2, IgG 3, orIgG4, and for example, they may be from a human the immunoglobulin(antibody).

The anti-Ang2 antibody may be a monoclonal antibody. The monoclonalantibody may be prepared by methods well known in the art. For example,it may be prepared using a phage display technique. Alternately, theAng2 antibody may be prepared into a mouse-derived monoclonal antibodyby methods set forth in the paper written by Schwaber, et al (Schwaber,J and Cohen, E. P., “Human×Mouse Somatic Cell Hybrid Clones SecretingImmunoglobulins of Both Parental Types,” Nature, 244 (1973), 444-447).

Individual monoclonal antibodies may be screened using a typical ELISA(Enzyme-Linked ImmunoSorbent Assay) format, based on the bindingpotential with Ang2. Inhibitory activities can be verified throughfunctional analysis such as competitive ELISA for verifying themolecular interaction of binding assemblies or functional analysis suchas a cell-based assay. Then, with regard to monoclonal antibody membersselected on the basis of their strong inhibitory activities, theiraffinities (Kd values) to Ang2 may be each verified.

The remaining portions of the anti-Ang2 antibody not including theantigen binding portions of the finally selected antibodies may beprepared as not only human immunoglobulin antibodies but also humanizedantibodies. Preparation of humanized antibodies is well known in the art(Almagro, J. C. and Fransson, J., “Humanization of antibodies,”Frontiers in Bioscience, 13(2008), 1619-1633).

Another embodiment provides a hybridoma cell line which produces amonoclonal antibody of the anti-Ang2 antibody. The hybridoma cell linemay be a cell line having accession number (KCLRF-BP-00295).

A polypeptide comprising an amino acid sequence represented by SEQ IDNO: 20, for example, an amino acid sequence of SEQ ID NO: 14 or SEQ IDNO: 15, may be useful as a CDR-H2 of an affinity-matured anti-Ang2antibody or antigen-binding fragment thereof. In addition, a polypeptidecomprising an amino acid sequence represented by SEQ ID NO: 21, forexample, an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17, maybe useful as a CDR-L1 of an affinity-matured anti-Ang2 antibody orantigen-binding fragment thereof. In addition, a polypeptide comprisingan amino acid sequence represented by SEQ ID NO: 22, for example, anamino acid sequence of SEQ ID NO: 18, may be useful as a CDR-L2 of anaffinity-matured anti-Ang2 antibody or antigen-binding fragment thereof.In addition, a polypeptide comprising an amino acid sequence representedby SEQ ID NO: 23, for example, an amino acid sequence of SEQ ID NO: 19,may be useful as a CDR-L3 of an affinity-matured anti-Ang2 antibody orantigen-binding fragment thereof.

Therefore, an embodiment provides a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 20 (e.g., SEQID NO: 14 or SEQ ID NO: 15), SEQ ID NO: 21 (e.g., SEQ ID NO: 16 or SEQID NO: 17), SEQ ID NO: 22 (e.g., SEQ ID NO: 18), and SEQ ID NO: 23(e.g., SEQ ID NO: 19). As described above, the polypeptide may be usefulas a complementarity determining region of an affinity-matured anti-Ang2antibody or antigen-binding fragment thereof.

Another embodiment provides a polynucleotide encoding the polypeptide.Another embodiment provides a recombinant vector comprising (orcarrying) the polynucleotide. Another embodiment provides a recombinantcell comprising (or transfected with) the recombinant vector.

Another embodiment provides a complex of Ang2 and a humanized and/oraffinity-matured anti-Ang2 antibody in which the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof and Ang2 are bound to each other.

Another embodiment provides a composition for inducing binding of Ang2with a Tie2 receptor, including the complex of Ang2 and an anti-Ang2antibody as an active ingredient. Another embodiment provides a methodfor inducing binding of Ang2 with a Tie2 receptor, includingadministering the complex of Ang2 and a humanized and/oraffinity-matured anti-Ang2 antibody to a subject. The subject may be inneed of binding between Ang2 and Tie2 receptor. The method for inducingbinding of Ang2 with Tie2 receptor may further include a step ofidentifying a subject who is in need of binding between Ang2 and Tie2receptor, prior to the administration step. Another embodiment providesa composition for activating a Tie2 receptor including the complex ofAng2 and a humanized and/or affinity-matured anti-Ang2 antibody as anactive ingredient.

Another embodiment provides a method for activating a Tie2 receptorincluding administering the complex of Ang2 and a humanized and/oraffinity-matured anti-Ang2 antibody to a subject. The subject may be inneed of activating the Tie2 receptor. The Tie2 receptor activationmethod may further include a step of identifying a subject who is inneed of activating the Tie2 receptor, prior to the administration step.The subject may be selected from mammals including primates such ashumans and monkeys or rodents such as rats and mice, or cells, tissues,or body fluids (e.g., blood, serum, etc.) isolated therefrom orartificially cultured. Another embodiment provides a use of the complexof Ang2 and a humanized and/or affinity-matured anti-Ang2 antibody ininducing binding of Ang2 with a Tie2 receptor and/or activating a Tie2receptor.

As described above, since the humanized and/or affinity-maturedanti-Ang2 antibody or an antigen-binding fragment thereof has a functionof inhibiting abnormal angiogenesis by inhibiting the functions of Ang2,it is applicable to prevent, alleviate, improve, and/or treat variousdiseases (e.g., cancer) related to abnormal angiogenesis. Moreover,since the humanized and/or affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof does not inhibit binding between Ang2and Tie2, it can activate a Tie2 signaling by activating Tie2, and itaccelerates the formation of vascular endothelium or lymphaticendothelium and increases mobility, to suppress vascular permeabilityincrease, whereby it is applicable to prevent, alleviate, improve,and/or treat various diseases related to vascular permeability (forexample, sepsis, eye disorders, etc.). Also, as described above, sincethe humanized and/or affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof accelerates the formation of vascularendothelium or lymphatic endothelium to increase the formation ofhealthy blood vessels and normalize the blood vessels, it is alsoapplicable to prevent, alleviate, improve, and/or treat various diseasesor symptoms requiring the formation of healthy blood vessels such aswound healing or ischemic disorders, and it reduces cancer growth andmetastasis possibility by changing the abnormally formed cancer bloodvessels into structurally and functionally normal forms. Moreover, theanti-Ang2 antibody or an antigen-binding fragment thereof has an effectof suppressing inflammatory response, whereby it is applicable toprevent, alleviate, improve, and/or treat various inflammatorydisorders. In addition, the anti-Ang2 antibody or an antigen-bindingfragment thereof has the effect of normalization of blood vessels incancer cells, thereby increasing the transporting efficiency of ananticancer agent through the normalized blood vessels into Ang2expressing cells. Therefore, when co-administered with an anticanceragent to a subject, the anti-Ang2 antibody or an antigen-bindingfragment thereof can be used as an adjuvant for enhancing sensitivenessto the anticancer agent and efficacy of the anticancer agent in thesubject.

The anticancer agent may be at least one agent selected from allchemical drugs, antibodies, genes (e.g., antisense oligonucleotide,siRNA, shRNA, microRNA, etc.), aptamer, cells for therapeutic use,agents for radiotherapy, and the like, for the use of growth inhibition,apoptosis promotion and metastasis inhibition of cancer cells or cancertissues. For example, the anticancer agent may be at least one selectedfrom the group consisting of cisplatin, carboplatin, oxalliplatin,paclitaxel, doxetaxel, vincristine, doxorubicin, daunorubicin,bleomycin, prednisone, methotrexate (MTX), 5-fluorouracil (5-FU),6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and the like, but not belimited thereto:

1) alkylating agents including i) platinum-based compounds includingcisplatin, carboplatin, oxaliplatin (oxliplatin), and the like, ii)nitrogen mustard-based compounds including mechlorethamine (nitrogenmustard), cyclophosphamide, ifosfamide, melphalan, chlorambucil, and thelike, iii) ethylenimine- and methylmelamine-based drugs includingthiotepa, altretamine, and the like, iv) methylhydrazine derivativesincluding procarbazine, and the like, v) alkyl sulfonate-based drugincluding busulfan, and the like, vi) nitrosourea-based drugs includingcarmustine, lomustine, and the like, and vii) triazine-based drugsincluding dacarbazine, and the like;

2) antimetabolites including i) pyrimidine derivatives includingfluorouracil (5-FU), capecitabine, cytarabine, gemcitabine, and thelike, ii) folic acid derivatives including methotrexate(MTX), and thelike, and iii) purine derivatives including mercaptopurine (6-MP), andthe like;

3) natural materials including i) vinca alkaloid including vinblastine,vincristine, vinorelvine, and the like, ii) taxane including paclitaxel,docetaxel, and the like, iii) epipodophyllotoxin including etoposide andthe like, and iii) camptothecin including topotecan, irinotecan, and thelike;

4) antibiotic materials including dactinomycin, doxorubicin,daunorubicin, mitomycin, bleomycin, and the like;

5) Targeted drugs including i) tyrosine kinase inhibitors includingimatinib, trastuzumab, cetuximab, gefitinib, erlotinib, and the like,and ii) angiogenesis inhibitors including bevacizumab, sunitinib,sorafenib, cabozantinib, pazopanib, regorafenib, vandetanib,ziv-afilibercept, and the like; and

6) prednisone, 6-thioguanine (6-TG), and the like.

Another embodiment provides a pharmaceutical composition including thehumanized and/or affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof as an active ingredient and a carrier.

Another embodiment provides a pharmaceutical composition for inhibitingangiogenesis including the humanized and/or affinity-matured anti-Ang2antibody or an antigen-binding fragment thereof as an active ingredient.Another embodiment provides a method for inhibiting angiogenesisincluding administering the humanized and/or affinity-matured anti-Ang2antibody or an antigen-binding fragment thereof to a subject. Thehumanized and/or affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof may be administered in amount that ispharmaceutically effective, which amount may be determined by theskilled medical practitioner or medical researcher. The subject may bein need of inhibiting angiogenesis. The angiogenesis inhibition methodmay further include a step of identifying a subject who is in need ofthe inhibition of angiogenesis (e.g., a subject with overexpression ofAng2), prior to the administration step. The step of identifying asubject may further comprise comparing a level of Ang2 in a biologicalsample (e.g., cell, tissue, fluid, etc.) from a subject to that of acontrol (a normal biological sample; e.g., with no overexpression ofAng2) and determining the subject as a suitable candidate for treatmentwith an anti-Ang2 antibody, when the level of Ang2 in a biologicalsample from the subject is higher than that of control.

Another embodiment provides a pharmaceutical composition for reducingvascular permeability including the humanized and/or affinity-maturedanti-Ang2 antibody or an antigen-binding fragment thereof as an activeingredient. Another embodiment provides a method for reducing vascularpermeability including administering the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof to a subject. The humanized and/or affinity-matured anti-Ang2antibody or an antigen-binding fragment thereof may be administered inamount that is pharmaceutically effective, which amount may bedetermined by the skilled medical practitioner or medical researcher.The subject may be in need of the reduction of vascular permeability.The vascular permeability reduction method may further include a step ofidentifying a subject who is in need of the reduction of vascularpermeability, prior to the administration step.

Another embodiment provides a pharmaceutical composition for preventingand/or treating a disease related to Ang2 overexpression, angiogenesis,and/or an increase in vascular permeability including the humanizedand/or affinity-matured anti-Ang2 antibody or an antigen-bindingfragment thereof as an active ingredient. Another embodiment provides amethod for preventing and/or treating a disease related to Ang2overexpression, angiogenesis, and/or an increase in vascularpermeability including administering the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof to a subject. The humanized and/or affinity-matured anti-Ang2antibody or an antigen-binding fragment thereof may be administered inamount that is pharmaceutically effective, which amount may bedetermined by the skilled medical practitioner or medical researcher.The subject may be in need of preventing and/or treating the diseaserelated to Ang2 overexpression, angiogenesis, and/or vascularpermeability increase. The prevention and/or treatment method mayfurther include a step of identifying a subject who is in need ofpreventing and/or treating a disease related to Ang2 overexpression,angiogenesis, and/or vascular permeability increase, prior to theadministration step. The subject may be one suffering from a diseaserelated to Ang2 overexpression, angiogenesis, and/or vascularpermeability increase.

Another embodiment provides a pharmaceutical composition for inducingnormal blood vessel formation including the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof as an active ingredient. Another embodiment provides a method ofinducing normal blood vessel formation, including administering thehumanized and/or affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof to a subject. The humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof may be administered in amount that is pharmaceuticallyeffective, which amount may be determined by the skilled medicalpractitioner or medical researcher. The subject may be in need ofinducing normal blood vessel formation. The method of inducing normalblood vessel formation may further include a step of identifying asubject who is in need of inducing normal blood vessel formation, priorto the administration step.

Another embodiment provides a pharmaceutical composition for preventingand/or treating a disease related to a decrease in normal blood vesselformation including the humanized and/or affinity-matured anti-Ang2antibody or an antigen-binding fragment thereof as an active ingredient.Another embodiment provides a method for preventing and/or treating adisease related to a decrease in normal blood vessel formation includingadministering the humanized and/or affinity-matured anti-Ang2 antibodyor an antigen-binding fragment thereof to a subject. The humanizedand/or affinity-matured anti-Ang2 antibody or an antigen-bindingfragment thereof may be administered in amount that is pharmaceuticallyeffective, which amount may be determined by the skilled medicalpractitioner or medical researcher. The subject may be in need ofpreventing and/or treating the disease related to a decrease in normalblood vessel formation. The prevention and/or treatment method mayfurther include a step of identifying a subject who is in need ofpreventing and/or treating a disease related to normal blood vesselformation decrease, prior to the administration step. The subject may beone suffering from a disease related to normal blood vessel formationdecrease.

Another embodiment provides a pharmaceutical composition for tissueregeneration and/or wound healing including the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof as an active ingredient. Another embodiment provides a methodfor tissue regeneration and/or wound healing including administering thehumanized and/or affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof to a subject. The humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof may be administered in amount that is pharmaceuticallyeffective, which amount may be determined by the skilled medicalpractitioner or medical researcher. The subject may be in need of tissueregeneration and/or wound healing. The method may further include a stepof identifying a subject who is in need of tissue regeneration and/orwound healing, prior to the administration step. A subject to whom theactive ingredient is administered may be a subject who has a skin tissueor organ tissue damage or has received a skin transplant.

Another embodiment provides a pharmaceutical composition for inhibitingAng2 and/or activating a Tie2 receptor including the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof as an active ingredient. Another embodiment provides a methodfor inhibiting Ang2 and/or activating a Tie2 receptor includingadministering the humanized and/or affinity-matured anti-Ang2 antibodyor an antigen-binding fragment thereof to a subject. The humanizedand/or affinity-matured anti-Ang2 antibody or an antigen-bindingfragment thereof may be administered in amount that is pharmaceuticallyeffective, which amount may be determined by the skilled medicalpractitioner or medical researcher. The subject may be in need of Ang2inhibition and/or Tie2 receptor activation. The Ang2 inhibition and/orTie2 receptor activation method may further include a step ofidentifying a subject who is in need of Ang2 inhibition and/or Tie2receptor activation, prior to the administration step. The anti-Ang2antibody or an antigen-binding fragment thereof may be in a form ofbeing bound to an antigen Ang2.

Another embodiment provides a pharmaceutical composition (an adjuvant)for enhancing the efficacy of an anticancer agent including thehumanized and/or affinity-matured anti-Ang2 antibody or anantigen-binding fragment thereof. Another embodiment provides a methodof enhancing the efficacy of an anticancer agent. The method includesadministering the humanized and/or affinity-matured anti-Ang2 antibodyor an antigen-binding fragment thereof to a subject in need ofadministration of the anticancer agent. The humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof may be administered in amount that is pharmaceuticallyeffective, which amount may be determined by the skilled medicalpractitioner or medical researcher. The method may further include,prior to the administration step, a step of identifying a subject inneed of administration of the anticancer agent. The enhancement of ananticancer agent may refer to making the anticancer agent exhibit moreexcellent anticancer effect at relatively low dosage, which is due tovascular normalization thereby increasing transporting efficiency of theanticancer agent into cancer tissues and increasing sensitiveness to theanticancer agent. The adjuvant may refer to a supplementarypharmaceutical composition used for enhancing the efficacy of ananticancer agent.

Since the function of the humanized and/or affinity-matured anti-Ang2antibody or an antigen-binding fragment thereof, which is an activeingredient of the above pharmaceutical compositions, is activated by thebinding with Ang2, the pharmaceutical compositions may further includeAng2 to enhance the function of the antibody or the antigen-bindingfragment thereof. The above methods may further include a step ofadministering Ang2 to a subject. The Ang2 may be administered in amountthat is pharmaceutically effective, which amount may be determined bythe skilled medical practitioner or medical researcher. The Ang2 may beadministered together with the anti-Ang2 antibody or an antigen-bindingfragment thereof simultaneously or sequentially in any order.

The pharmaceutical compositions described herein may further include apharmaceutically acceptable carrier, and the carrier may be thosecommonly used in the formulation of drugs, which may be one or moreselected from the group consisting of lactose, dextrose, sucrose,sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates,gelatin, calcium silicate, micro-crystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, and mineral oil, but is not limited thereto. Thepharmaceutical compositions may further include one or more selectedfrom the group consisting of a diluent, an excipient, a lubricant, awetting agent, a sweetener, a flavor enhancer, an emulsifying agent, asuspension agent, and a preservative.

Pharmaceutically effective amounts of the pharmaceutical compositions,or the antibody or the antigen-binding fragment thereof may beadministered orally or parenterally. The parenteral administration mayinclude intravenous injection, subcutaneous injection, muscularinjection, intraperitoneal injection, endothelial administration, localadministration, intranasal administration, intrapulmonaryadministration, and rectal administration. Since oral administrationleads to digestion of proteins or peptides, an active ingredient in thecompositions for oral administration must be coated or formulated toprevent digestion in stomach. In addition, the composition may beadministered using an optional device that enables an active substanceto be delivered to target cells.

The content of the anti-Ang2 antibody or an antigen-binding fragmentthereof in the pharmaceutical compositions may be prescribed in avariety of ways, depending on factors such as formulation methods,administration methods, age of subjects, body weight, gender, pathologicconditions, diets, administration time, administration interval,administration route, excretion speed, and reaction sensitivity. Forexample, a daily dosage of the anti-Ang2 antibody or an antigen-bindingfragment thereof may be within the range of 0.001 to 1000 mg/kg,particularly 0.01 to 100 mg/kg, and more particularly 0.1 to 50 mg/kg,but is not limited thereto. The daily dosage may be formulated into asingle formulation in a unit dosage form or formulated in suitablydivided dosage forms, or it may be manufactured to be contained in amultiple dosage container. The term “pharmaceutically effective amount”as used herein refers to a content or dose of an active ingredientcapable of showing desirable pharmacological effects and it may bedetermined in a variety of ways, depending on factors such asformulation methods, administration methods, age of subjects, bodyweight, gender, pathologic conditions, diets, administration time,administration interval, administration route, excretion speed, andreaction sensitivity.

The pharmaceutical compositions may be formulated into a form of asolution in oil or an aqueous medium, a suspension, syrup, anemulsifying solution, an extract, powder, granules, a tablet, or acapsule, and may further include a dispersing or a stabilizing agent forthe formulation.

In particular, the pharmaceutical compositions including the anti-Ang2antibody or an antigen-binding fragment thereof may be formulated intoan immunoliposome since it contains an antibody or an antigen-bindingfragment. A liposome containing an antibody may be prepared using anymethods widely known in the art. The immunoliposome may be a lipidcomposition including phosphatidylcholine, cholesterol, andpolyethyleneglycol-derivatized phosphatidylethanolamine, and may beprepared by a reverse phase evaporation method. For example, Fab′fragments of an antibody may be conjugated to the liposome through adisulfide-exchange reaction.

Meanwhile, as the humanized and/or affinity-matured anti-Ang2 antibodyor an antigen-binding fragment thereof specifically binds to Ang2, thiscan be used to detect Ang2, and the presence of the overexpression ofAng2 can be verified through it. Accordingly, another embodimentprovides a composition for detecting Ang2 and a composition fordiagnosing a disease related to Ang2 overexpression, angiogenesis, anincrease in vascular permeability, and/or a decrease in normal bloodvessel formation, including the humanized and/or affinity-maturedanti-Ang2 antibody or an antigen-binding fragment thereof.

In another embodiment, provided is a method for detecting Ang2 includingtreating (or contacting) a biological specimen obtained (or isolated)from a subject with the humanized and/or affinity-matured anti-Ang2antibody or an antigen-binding fragment thereof; and identifying thepresence of an antigen-antibody reaction between Ang2 and the anti-Ang2antibody or fragment thereof. Also, there is provided a method ofdiagnosing a disease related to Ang2 overexpression, angiogenesis, anincrease in vascular permeability, and/or a decrease in normal bloodvessel formation, including treating (or contacting) a biologicalspecimen obtained from a subject with the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof; and identifying the presence of an antigen-antibody reactionbetween Ang2 and the anti-Ang2 antibody or fragment thereof, wherein asubject is determined to have the disease related to Ang2overexpression, angiogenesis, and/or an increase in vascularpermeability, when the antigen-antibody reaction is present, or isincreased compared to the antigen antibody reaction of a control samplethat represents known “normal” levels of antigen-antibody reaction(e.g., the control sample may be an animal such as human, or cells ortissues obtained therefrom, who has no disease related to Ang2overexpression, angiogenesis, and/or an increase in vascularpermeability).

In the method of the diagnosing, a subject may be determined to haveAng2 overexpression symptoms, or have the diseases when anantigen-antibody reaction is detected in the step of identifying thepresence of an antigen-antibody reaction. Therefore, method may furthercomprise determining a subject to have Ang2 overexpression symptoms, orhave Ang2 overexpression related diseases when an antigen-antibodyreaction is detected in the step of identifying, after the step ofidentifying the presence of an antigen-antibody reaction, and wherein asubject is determined to have the disease related to Ang2overexpression, when the antigen-antibody reaction is present, or isincreased compared to the antigen antibody reaction of a control samplethat represents known “normal” levels of antigen-antibody reaction(e.g., the control sample may be an animal such as human, or cells ortissues obtained therefrom, who has no disease related to Ang2overexpression).

The biological specimen may be selected from the group consisting ofcells, tissues and body fluids obtained (isolated) from a subject.

The step of identifying the presence of the antigen-antibody reactionmay be performed using various methods known in the art. For example, itmay be measured through an ordinary enzyme reaction, fluorescence,luminescence, and/or radioactivity detection and particularly, it may bemeasured by a method selected from the group consisting ofimmunochromatography, immunohistochemistry, enzyme linked immunosorbentassay (ELISA), radioimmunoassay (MA), enzyme immunoassay (EIA),fluorescence immunoassay (FIA), luminescence immunoassay (LIA), westernblotting, etc., but is not limited thereto.

The subjects which the pharmaceutical composition or the antibody or theantigen-binding fragment thereof is administered to or is aimed todiagnose may be mammals including primates such as humans and monkeys,or rodents such as rats and mice, or cells, tissues and body fluidsisolated therefrom or artificially cultured.

The diseases related to angiogenesis and/or an increase in vascularpermeability and/or Ang2 overexpression may be cancer; cancermetastasis; ocular blood vessel disorders such as retinopathy ofprematurity, macular degeneration (e.g., age-related maculardegeneration), diabetic retinopathy, neovascular glaucoma, etc.;inflammatory disorders such as psoriasis, asthma, rheumatoid arthritis,pneumonia, chronic inflammation, etc.; infectious disorders (infection);cardiovascular disorders such as hypertension, arteriosclerosis, etc.;renal disease; sepsis; asthma; edema; hereditary hemorrhagictelangiectasia (HHT), etc. The cancer may be those overexpressing Ang2,it may be a solid cancer or a blood cancer, and it may be, but notlimited to, selected from the group consisting of squamous cellcarcinoma, small-cell lung cancer, non-small-cell lung cancer,adenocarcinoma of the lung, squamous cell carcinoma of the lung,peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball,rectal cancer, cancer near the anus, esophagus cancer, small intestinaltumor, endocrine gland cancer, parathyroid cancer, adrenal cancer,soft-tissue sarcoma, urethral cancer, chronic or acute leukemia,lymphocytic lymphoma, hepatocellular cancer, gastric cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, large intestinecancer, endometrial carcinoma or uterine carcinoma, salivary glandtumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer,head or neck cancer, brain cancer, osteosarcoma, etc. The cancer may bea primary cancer or a metastatic cancer.

The diseases related to a decrease in normal blood vessel formation arediseases that require the induction of normal blood vessel formation andmay be selected from the group consisting of ischemic disorders such asmyocardial infarction, angina, cerebral infarction, stroke (ischemicstroke), etc., Buerger's disease (thromboangiitis obliterans), avascularnecrosis, foot ulcer (e.g., diabetic foot ulcer), erectile dysfunctionand so on.

Another embodiment provides a complex in which the humanized and/oraffinity-matured anti-Ang2 antibody or an antigen-binding fragmentthereof, Ang2, and Tie2 receptor are bound (i.e., the anti-Ang2 antibodyor an antigen-binding fragment thereof binds to Ang2, Ang2 binds to Tie2receptor, and the anti-Ang2 antibody or an antigen-binding fragmentthereof and Tie2 receptor are bound via Ang2). The complex may bepresent inside the body or present in a cell isolated from the body.Also, the antibody in the complex may form a dimer with another antibodyin an adjacent complex, thereby clustering two or more complexes, toform a cluster including two or more complexes. Such action is relatedto the Tie2 receptor activation function of the anti-Ang2 antibody. Thecomplex can be used to monitor the action of the anti-Ang2 antibody,i.e., the presence of Ang2 inhibition and/or Tie2 receptor activation,or simply used to inhibit Ang2 and/or to activate Tie2 receptor.

Previously, there were attempts to inhibit angiogenesis by Ang2 byinhibiting binding between Ang2 and its receptor Tie2, but an antibodywhich specifically binds to Ang2 to induce the intracellularinternalization and degradation of Ang2 and at the same time, maintainsa binding ability with Ang2 and Tie2 receptor to form anantibody-Ang2-Tie2 receptor complex, thereby activating the Tie2receptor was not known. Thus, the invention proposes a humanizationand/or affinity maturation of an antibody capable of inhibitingangiogenesis by Ang2 and reducing vascular permeability by providing anantibody which inhibits Ang2 and at the same time activates the Tie2receptor to accelerate its downstream signaling. Also, the humanizedand/or affinity-matured antibody proposed in the invention isanticipated to be applicable to diagnose and treat abnormal blood vesselformation-related disorders other than cancer and/or disorders caused byvascular permeability increase. The humanized and/or affinity-maturedantibody can be utilized for combination therapy with chemical medicinesand other anticancer drugs, and is expected to be employed for antibodyfragments, bi- or multi-specific antibodies, protein scaffolds, etc.using Ang2 specific recognition activity.

EXAMPLES

Hereafter, the present invention will be described in detail byexamples.

The following examples are intended merely to illustrate the inventionand are not construed to restrict the invention.

Example 1. Preparation of a Mouse Anti-Ang2 Antibody, 10D6

1.1. Immunization of a Mouse

A human Ang2 protein (R&D systems; 623-AN-025/CF) was administered to5-week-old BALB/c mice along with an adjuvant to induce an immuneresponse and then, hybridomas that produce an individual anti-Ang2antibody were prepared according to the known methods described in thepaper written by Schwaber, et al (Schwaber, J and Cohen, E. P.,“Human×Mouse Somatic Cell Hybrid Clones Secreting Immunoglobulins ofBoth Parental Types,” Nature, 244 (1973), 444-447).

More specifically, to obtain immunized mice necessary for developinghybridoma cell lines, 100 μg (microgram) of human Ang2 protein (R&DSystems) mixed with the same amount of a complete Freund's adjuvant wasadministered via an intraperitoneal injection to each of five4˜6-week-old BALB/c mice (Japan SLC, Inc.). After two weeks, the antigen(half the previously injected amount) mixed with an incomplete Freund'sadjuvant using the same method as described above was administered toeach mouse via an intraperitoneal injection. After one additional week,a final boosting was performed and three days later, blood was collectedfrom the tail of each mouse to obtain serum, which was then diluted at1/1000 with PBS and subjected to an ELISA to verify that the titer of anantibody recognizing Ang2 was increased. From the results, mice in whicha sufficient amount of the antibody was obtained were selected, and acell fusion process was performed on the selected mice.

Three days before the cell fusion experiment, a mixture of 50 μg of PBSand 100 μg of human Ang2 protein (R&D systems) was administered via anintraperitoneal injection to BALB/c mice (Japan SLC, Inc.), and aftereach immunized mouse was anesthetized, its spleen located on the leftside of the body was extracted. The extracted spleen was ground with amesh to isolate cells, which were mixed with a culture medium (DMEM,Hyclon) to prepare a spleen cell suspension. The suspension wascentrifuged to collect a cell layer. The obtained 1×10⁸ spleen cellswere mixed with 1×10⁷ myeloma cells (Sp2/0), and the mixture wascentrifuged to precipitate the cells. The centrifuged precipitate wasslowly dispersed, treated with 1 ml of 45% polyethylene glycol (PEG1500) contained in a culture medium (DMEM), and maintained at 37° C. forone minute before adding 1 ml of a culture medium (DMEM). Subsequently,10 ml of the culture medium (DMEM) was added for 1 minute to theresultant, which was incubated in a water bath at 37° C. for 5 minutesand then re-centrifuged after the total volume was adjusted to 50 ml.The resulting cell precipitate was re-suspended in an isolation medium(HAT medium) at a concentration of 1˜2×10⁵/ml, and the resultantsuspension was distributed at 0.1 ml to the each well of a 96-wellplate, which was then incubated in a carbon dioxide incubator at 37° C.to prepare the hybridoma cell groups.

1.2. Production and Purification of a Monoclonal Antibody

The above obtained individual antibody producing hybridomas werescreened using a typical ELISA format to select hybridomas which produce95 anti-Ang2 monoclonal antibodies among the hybridomas differentiatedfrom their mother hybridomas, based on their binding potential withAng2.

More specifically, to select the hybridoma cells that specifically reactonly to Ang2 protein among the hybridoma cell groups prepared in Example1.1 above, an ELISA assay method using a human Ang2 protein as anantigen was used for screening.

Human Ang2 protein was added at 100 ng per each well to a microtiterplate to be adhered to the surface of the plate, and unreacted antigenswere removed by washing. 50 microliters of the hybridoma cell cultureobtained in Example 1 above was added to each well to react for 1 hourand then, the wells were sufficiently washed with phosphate bufferedsaline-TWEEN 20 (PBST) solution to remove unreacted culture solution.Goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP) wasadded thereto, a reaction was allowed to occur at a room temperature for1 hour and then, washing was sufficiently performed with the TBSTsolution. Subsequently, substrate solution (OPD) of peroxidase was addedto each well to react, and the reaction degree was measured by theabsorption at 450 nm using an ELISA reader to repeatedly selecthybridoma cell lines that secret antibodies having specifically highbinding affinity only to human Ang2 protein. A limiting dilution wasperformed on the hybridoma cell lines obtained through repetitiveselection to obtain final 58 clones of hybridoma cell lines producingmonoclonal antibodies.

Each hybridoma obtained above was cultured in DMEM (Dulbeco's ModifiedEagle's Medium) and then, the culture solutions were collected andsubjected to Protein G-affinity chromatography method to purifyanti-Ang2 monoclonal antibodies produced from each hybridoma.

First, the hybridoma cells cultured in 50 ml of culture medium (DMEM)containing 10% (v/v) FBS were centrifuged to obtain a cell precipitate,which was washed at least twice with 20 ml of PBS to remove the FBS. Thecell precipitate was re-suspended in 50 ml of the culture medium (DMEM)and then incubated in a carbon dioxide incubator at 37° C. for 3 days.Subsequently, the cell culture was centrifuged to remove theantibody-producing cells, and the culture medium including the secretedantibodies was isolated and then, stored at 4° C. or used directly.Antibodies were purified from 50 to 300 ml of the culture medium usingan AKTA purification device (GE Healthcare) equipped with an affinitycolumn (protein G agarose column; Pharmacia, USA). The purifiedantibodies were stored for subsequent use after replacing thesupernatant with PBS using a filter for protein aggregation (Amicon),and used for the following examples.

1.3. Examination of Functions of Monoclonal Antibodies and Selection ofMouse Anti-Ang2 Antibody 10D6

As Ang2 induces a change in vascular endothelial cells by binding to aTie-2 receptor expressed in the vascular endothelial cells to induce thephosphorylation of the receptor and activate it, a test for analyzing aninfluence of the anti-Ang2 antibody on Tie2 phosphorylation wasconducted using a cell-based assay.

For this, HUVEC (ATCC) cells (1×10⁵ cells) were cultured in a 100 mmculture dish using EGM-2 (Lonza) media at 37° C. and when they reached80˜90% confluency, the media were replaced with serum-free medium(Lonza) and cultured at 37° C. for 6 to 16 hours. The dish was washedonce with PBS and after the replacement with 1 nM sodium orthovanadate(Sigma)-mixed serum free media (Lonza), they were further cultured for10 min. After washed once again with PBS, the cultured cells weretreated with a mixture prepared by mixing the anti-Ang2 antibody (10D6)having various concentrations (600˜0.06 nM) with 40 nM of Ang2 protein(R&D systems) and letting them stand for 20 min. and further culturedfor 10 min.

The cells were washed using PBS, treated with 400 μl of a lysis buffer(Roche), collected to a tube to be dissolved at 4° C. for 30 min. andthen, centrifuged at 13,000 rpm for 15 min. to measure a supernatantusing Nanodrop. 1 μg of Tie2 antibody (R&D system) was added to 0.8 mgof a cell lysate, which was then overnight reacted at 4° C. and thensubjected to immunoprecipitation by the addition of protein A bead (GEHealthcare) thereto. The thus obtained reactant was centrifuged at13,000 rpm for 15 min. to obtain a pellet, which was washed two to threetimes with a lysis buffer (Roche), added to a sample buffer (Invitrogen)mixed with a reducing agent, and boiled at 95° C. for 5 min., and then,applied to NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and transferredonto Nitrocellulose membrane (Invitrogen).

To examine the presence of the phosphorylation of Tie2, the membraneswere blocked with PBST mixed with 3% (v/v) skim milk (Sigma) for 30 min.and identified using an HRP-conjugated anti-phospho tyrosine antibody(Millipore). For Tie2 identification, the blots were reacted in astripping buffer (Thermo) for 15 min, then blocked again and identifiedusing an anti-Tie2 antibody (Santa cruz). An antibody, which shows moreintensive ability to induce a phosphorylation of Tie2 receptor when itis added together with Ang2 at the concentration of 60 nM, compared tothe case treated with Ang2 only, was selected and named as 10D6.

The hybridoma producing 10D6 was deposited in the Korean Cell Line Banklocated at Yongon-dong, Chongno-gu, Seoul, South Korea, as of Apr. 23,2013 and received accession number KCLRF-BP-00295.

1.4. Analysis of Binding Affinity of Mouse Antibody 10D6 to Ang2

The binding affinity of the above antibody to human Ang2 protein wasmeasured by an surface plasmon resonance (SPR) method using a BIAcoreT100 (GE Healthcare). The SPR method uses refractive index change oflight which passes a sensor chip according to the state of materialscoated onto the sensor chip, and if an antigen or an antibody is flowedonto a chip coated with the antigen or antibody, it causes changes inrefractive index due to their binding and Kd values are thus calculatedfrom the measured values.

First, anti-His antibody was immobilized on a CM5 sensor chip (GEhealthcare) up to 8,000 RU levels using a pH 5.0 acetate solution and anamine coupling kit (GE Healthcare). 6 μg/ml of a recombinant hAng2(C-His, R&D Systems) protein was flowed onto the chip to be captured at100 to 200 RU levels. The antibody obtained in Example 2 above wasdiluted serially to twice each time starting from 100 nM concentrationand it was each flowed onto the chip to allow it to be bound to (on),dissociated from (off), and regenerated (using 10 mM NaOH solution) fromthe antigen captured on the sensor chip, thereby to measureantigen-antibody affinity. With regard to hAng2, such experiments wereconducted, and the results are as shown in the following Table 4.

TABLE 4 antibody hAng2 (Kd) SAIT-ANG2-AB-m10D6 8.0 nM

1.5. ELISA Assay for Identifying Formation of 10D6-Ang2-Tie2 Complex

As it was confirmed that 10D6 anti-Ang2 antibody activates Tie2signaling without inhibiting Ang2-Tie2 binding, an ELISA was conductedto see whether a complex between the antibody and Ang2:Tie2 receptor isformed.

A 96-well MaxiSorp™ flat-bottom plate (Nunc) was coated with 4 μg/ml ofTie2-Fc (R&D systems) or BSA (Sigma). Then, the plate was washed fivetimes with 0.05% (v/v) Tween-20-containing PBS (Phosphate Buffer Saline)and blocked with 1% (v/v) BSA (Bovine serum albumin; Sigma)-containingPBS at a room temperature for 2 hours. 0.25 μg/ml of Ang2 and 2 μg/ml of10D6 antibody were added to each well of the plate, which was allowed toreact at a room temperature for 2 hours and then washed five times withPBST. After that, an anti-mouse IgG antibody (Sigma) conjugated with HRPdiluted in 1% (v/v) BSA-containing PBS at a ratio of 1:5,000 (v/v) wasadded in an amount of 100 μl to each well to react at a room temperaturefor 1 hour and then, the plate was washed five times with PBST. Lastly,100 μl (microliter) of TMB substrate (Cell Signaling) was added to eachwell of the plate to induce color development for 3 min. and then, thereaction was ceased by the addition of 100 μl of Stop solution (CellSignaling) and OD450 values were measured on a plate reader (MolecularDevices).

The thus obtained results are shown in FIG. 6. As seen in FIG. 6, it wasconfirmed that 10D6 antibody formed a complex by binding to Ang2 whichwas bound to Tie2.

1.6. Competition ELISA Assay of Mouse Antibody 10D6 to Ang2-Tie2 Binding

Ang2-Tie2 binding competition ELISA was conducted using the antibodybinding to Ang2 prepared in Example 1.3 above.

More specifically, MaxiSorp™ flat-bottom plate (Nunc) of 96-well wascoated with hTie2-Fc (R&D Systems) which is a protein bound with 4 μg(microgram)/ml of Fc of human IgG1. After that, the plate was washedfive times with 0.05% (v/v) Tween-20-containing PBS (phosphate buffersaline) and then blocked with 1% (v/v) BSA (bovine serum albumin;Sigma)-containing PBS at a room temperature for 2 hour.

For Ang2:Tie2 competition ELISA, each anti-Ang2 antibody obtained inExample 2 was placed at various concentrations of 400 nM to 0.001 nMinto each well coated with the hTie-2/Fc fusion protein along with 1%(v/v) BSA and 400 ng/ml of a FLAG-tagged hAng2 and then, the plate wasallowed to react at a room temperature for 2 hours and washed five timeswith PBST. After that, an anti-FLAG antibody (Sigma) conjugated with HRPdiluted in 1% (v/v) BSA-containing PBS at a ratio of 1:5,000 (v/v) wasadded in an amount of 100 μl (microliter) to each well to react at aroom temperature for 1 hour and then, the plate was washed five timeswith PB ST. Lastly, 100 μl (microliter) of TMB substrate (CellSignaling) was added to each well of the plate to induce colordevelopment for 3 min. and then, the reaction was ceased by the additionof 100 μl of Stop solution (Cell Signaling) and OD450 values weremeasured on a plate reader (Molecular Devices).

For comparison, the same test was carried out using 4H10 which is ananti-Ang2 antibody inhibiting Ang2-Tie2 binding. The 4H10 is an antibodyhaving the following heavy chain variable region and light chainvariable region.

Heavy chain variable region (SEQ ID NO: 12):EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSLISPDSSSIVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDL ISFWRGGFDYWGQGTLVTVSSLight chain variable region (SEQ ID NO: 13)QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVNWYQQLPGTAPKLLIYADSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSGYV FGGGTKLTVLG

An inhibitory degree (%) against Ang2-Tie2 binding is shown in FIG. 1.As seen in FIG. 1, unlike 4H10 which is an anti-Ang2 antibody inhibitingAng2-Tie2 binding, antibody 10D6 did not inhibit binding betweenAng2-Tie2 receptor.

Example 2: Gene Cloning of Mouse Antibody 10D6

A whole RNA was obtained using RNeasy mini kit (Qiagen) from theantibody-producing hybridoma (2×10⁶ cells) obtained from Example 1.3above. Then, by using this as a template, only the gene sequence of theheavy chain and light chain variable regions of the monoclonal antibodyto be produced in the hybridoma was amplified using a OneStep RT-PCR kit(Qiagen), a Mouse Ig-Primer Set (Novagen), and a thermocycler (GeneAmpPCR System 9700, Applied Biosystem) under the following conditions: 5min. at 94° C.; [30 min. at 50° C., 15 min. at 95° C.], [1 min. at 94°C., 1 min. at 50° C., 2 min. at 72° C.]×35 cycles; 6 min. at 72° C.;cooling to 4° C.

The PCR products obtained from each reaction were subjected to a directDNA sequencing to obtain the amino acid sequences of the CDR, heavychain variable regions and light chain variable regions of the antibody,and nucleotide sequences encoding them, and the obtained results are setforth in the following Tables 5 to 8.

TABLE 5 Amino acid sequence of heavy chain CDR Antibody CDRH1-KABATCDRH2-KABAT CDRH3-KABAT SAIT-ANG2-AB-m10D6 SDYAWN YINYSGNTDYNPSLKSGNFEGAMDY (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3)

TABLE 6 Amino acid sequence of light chain CDR Antibody CDRL1-KABATCDRL2-KABAT CDRL3-KABAT SAIT-ANG2-AB-m10D6 KASQSVSNDVA YASNRYP QQDYSSPWT(SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6)

TABLE 7 Antibody Sequence of heavy chain variable regionSAIT-ANG2-AB-m10D6 DVQLQESGPDLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKL EWMGYINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTA TYYCAR GNFEGAMDYWGQGTSVTVSS (SEQ ID NO: 7) GATGTGCAGCTTCAGGAGTCGGGACCTGACCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAACTACAGTGGTAACACTGACTACAACCCATCTCTCAAAAGTCGAAGCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGGGGACACAGCCACATATTACTGTGCAAGAGGTAACTTCGAAGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 8)

TABLE 8 Antibody Sequence of light chain variable regionSAIT-ANG2-AB-m10D6 SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA WYQQKPGQSPK LLIYYASNRYP GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC QQD YSSPWTFGGGTKLEIK (SEQ ID NO: 9)agtattgtgatgacccagactcccaaattcctgcttgtatcagcaggagacagggttaccataacctgcaaggccagtcagagtgtgagtaatgatgtagcttggtaccaacagaagccagggcagtctcctaaactgctgatatactatgcatccaatcgctaccctggagtccctgatcgcttcactggcagtggatatgggacggatttcactttcaccatcagcactgtgcaggctgaagacctggcagtttatttctgtcagcaggattatagctctccgtggacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID NO: 10)

(In above Tables 6 and 7, underlined bold letters are CDR1, CDR2, andCDR3 in sequence)

Based on the sequence information obtained above, single chain DNAsencoding the heavy chain variable region and the light chain variableregion, respectively, were prepared, and cloned into vectors comprisinga human kappa constant region coding gene and a CH1 region coding geneof human IgG1, respectively. In particular, a DNA fragment having theheavy chain variable region coding nucleotide sequence (SEQ ID NO: 8)was cloned into a vector of pOptiVEC™-TOPO TA Cloning Kit comprised inOptiCHO™ Antibody Express Kit (Cat no. 12762-019; Invitrogen), and a DNAfragment having the light chain variable region coding nucleotidesequence (SEQ ID NO: 10) was cloned into a vector of pcDNA™3.3-TOPO TACloning Kit(Cat no. 8300-01), using EcoRI(NEB, R0101S) and XhoI(NEB,R0146S), to construct a vector comprising the heavy chain variableregion and a vector comprising the light chain variable region forexpressing a chimeric antibody.

Example 3: Preparation of an scFv of Mouse Antibody 10D6

A gene for producing an scFv fragment using the heavy chain variableregion and the light chain variable region of mouse antibody 10D6 wasdesigned. The heavy chain variable region (amino acid sequence: SEQ IDNO: 7; coding nucleotide sequence: SEQ ID NO: 8) and the light chainvariable region (amino acid sequence: SEQ ID NO: 9; coding nucleotidesequence: SEQ ID NO: 10) were linked to form ‘VH-linker-VL’ construct,and the linker is designed to have the amino acid sequence of‘GGGGSGGGGSGGGGS(SEQ ID NO: 76)’. The amino acid sequence of thedesigned ‘VH-linker-VL’ (scFv of 10D6) is represented in SEQ ID NO: 80and the coding nucleotide sequence thereof is represented in SEQ ID NO:81.

Example 4: Preparation of Gene Library for Affinity Maturation

4.1. Selection of Target CDR and Preparation of Primers

To perform affinity maturation, six complementary determining regions(CDRs) were defined from the prepared mouse antibody 10D6 according tothe ‘Kabat numbering’ rule. The CDRs are summarized in Table 9:

TABLE 9 CDR Amino acid sequence CDR-H1 SDYAWN (SEQ ID NO: 1) CDR-H2YINYSGNTDYNPSLKS (SEQ ID NO: 2) CDR-H3 GNFEGAMDY (SEQ ID NO: 3) CDR-L1KASQSVSNDVA (SEQ ID NO: 4) CDR-L2 YASNRYP (SEQ ID NO: 5) CDR-L3QQDYSSPWT (SEQ ID NO: 6)

For use in the introduction of random sequences into the CDRs of theantibody, primers were designed as follows. Conventionally, N codonswere utilized to introduce bases at the same ratio (25% A, 25% G, 25% C,25% T) into desired sites of mutation. In this experiment, theintroduction of random bases into the CDRs of 10D6 was conducted in sucha manner that, of the three nucleotides per codon in the wild-typepolynucleotide encoding each CDR, the first and second nucleotidesconserved over 85% of the entire sequence while the other threenucleotides were introduced at the same percentage (each 5%) and thatthe same possibility was imparted to the third nucleotide (33% G, 33% C,33% T).

4.2. Construction of Gene Library of scFv of 10D6 Antibody

The construction of antibody gene libraries through the introduction ofrandom sequences was carried out using the primers synthesized in thesame manner as in Example 4.1. Two PCR products were obtained using apolynucleotide covering the 10D6 scFv (SEQ ID NO: 81) as a template (seeFIG. 3), and were subjected to overlap extension PCR to give scFvlibrary genes for 10D6 antibodies in which only desired CDRs weremutated. 10⁷˜10⁸ libraries targeting each of the six CDRs prepared fromthe scFv library genes were constructed.

The affinity for Ang2 of each library was compared to that of thewild-type. Most libraries were lower in affinity for Ang2, compared tothe wild-type. However, in some mutants, the affinity for Ang2 wasretained.

Example 5: Selection of Antibody with Improved Affinity from Libraries

Among the scFv libraries provided in Example 4, the scFv fragmentsshowing upper 1.0 percent of affinity to Ang-2 were selected, and thisprocess was repeated four times. The nucleotide sequence of each of theselected scFv was analyzed. The obtained nucleotide sequences aresummarized in Table 10, and were converted into IgG forms (a heavy chainconstant region: constant region of human IgG1, a light chain constantregion: constant region of human KAPPA Chain). Five antibodies whichwere respectively produced from clones VH-6.6, VH-6.7, VL-(6.11),VL-(6.17), and VL-HU1(6.22) were used in the subsequent experiments.

TABLE 10 Library clones constructed CDR sequence VH-6.6 CDR-H2KISYSGKTDYNPSLKS (SEQ ID NO: 14) VH-6.7 CDR-H2 KINYAGNTDYNPSLKS(SEQ ID NO: 15) VL-(6.11) CDR-L1 KASQSVSNDVH (SEQ ID NO: 16) VL-(6.17)CDR-L3 QHDYSSPFT (SEQ ID NO: 19) VL-(6.22) CDR-L1 +KASQSVSNDVH (SEQ ID NO: 16) + CDR-L3 QHDYSSPFT (SEQ ID NO: 19)

Example 6: Preparation of Humanized Antibody 10D6-HU1, 10D6-HU2,10D6-HU3, and 10D6-HU5, from Mouse Antibody 10D6

6.1. Heavy Chain Humanization

To design three domains 10D6-HU1 Heavy, 10D6-HU2-heavy, and10D6-HU5-heavy, human germline genes which share the highestidentity/homology with the VH gene of the mouse antibody 10D6 purifiedwere analyzed through an Ig BLAST(http://www.ncbi.nlm.nih.gov/igblast/). The analysis results revealedthat IGHV4-b*01 (DP-67; accession number: Z12367) has anidentity/identity/homology of 72% at the amino acid level. CDR-H1(SEQ IDNO: 1), CDR-H2(SEQ ID NO: 2), and CDR-H3(SEQ ID NO: 3) of the mouseantibody 10D6 were defined according to Kabat numbering. A design wasmade to introduce the CDR of the mouse antibody 10D6 into the frameworkof IGHV4-b*01 (named as 10D6-HU1; SEQ ID NO: 77;QVQLQESGPGLVKPSETLSLTCAVSGYSISSDYAWNWIRQPPGKGLEWIGYINYSGNTDYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGNFEGAMDYWGQGTLVTVS S). Hereupon,a back mutation to the amino acid sequence of the mouse 10D6 wereconducted at positions 30 (S→T), to establish antibody 10D6-HU5 (SEQ IDNO: 56). Then, 10D6-HU5 was further mutated at positions 48 (I→M), 67(V→S), and 71 (V→R), to establish 10D6-HU2(SEQ ID NO: 78;QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQPPGKGLEWMGYINYSGNTDYNPSLKSRSTISRDTSKNQFSLKLSSVTAADTAVYYCARGNFEGAMDYWGQGTLVTV SS).

For use in designing 10D6-HU3-heavy, human antibody frameworks wereanalyzed by a BLAST search. The result revealed that the Herceptinbackbone, which known to show very low immunogenicity of about 0.1%level among the pre-existing humanized antibodies, is very similar inframework and sequence to the mouse antibody 10D6. CDR-H1, CDR-H2, andCDR-H3 of the mouse antibody 10D6 were defined according to Kabatnumbering and introduced into the Herceptin backbone to constructH4-heavy (SEQ ID NO: 42), wherein back mutations were conducted atpositions 27 (F-Y), 28(N→S), 30(K→T), 48(V→M), 49(A→G), 67 (F→S),71(A→R), 78(A→F), and 93(S→A), to establish 10D6-HU3(SEQ ID NO: 79;EVQLVESGGGLVQPGGSLRLSCAASGYSITSDYAWNWVRQAPGKGLEWMGYINYSGNTDYNPSLKSRSTISRDTSKNTFYLQMNSLRAEDTAVYYCARGNFEGAMDYWGQGTLVT VSS).

6.2. Light Chain Humanization

To design a H1-light, human germline genes which share the highestidentity/homology with the VL gene of the mouse antibody 10D6 wereanalyzed through an Ig BLAST (NCBI, Bethesday, Md.; available online at<<ncbi.nlm.nih.gov/igblast/>>). The analysis results revealedIGKV1-39*01(O12; accession number: X59315) has anidentity/identity/homology of 66% at the amino acid level. CDR-L1(SEQ IDNO: 4), CDR-L2(SEQ ID NO: 5), and CDR-L3(SEQ ID NO: 6) of the mouseantibody 10D6 were defined according to Kabat numbering. A design wasmade to introduce the CDR of the mouse antibody 10D6 into the frameworkof IGKV1-39*01.

Thereafter, DNA fragments of heavy chains (10D6-VHHU1, 10D6-VHHU2,10D6-VHHU3, and 10D6-VHHU5) were respectively cloned into a vector ofpOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody ExpressKit (Cat no. 12762-019, Invitrogen) using EcoRI(NEB, R0101S) andnheI(NEB, R0131), and a DNA fragment of a light chain (10D6-VLHU1(SEQ IDNO: 57), coding sequence: SEQ ID NO: 69) was cloned into a vector ofpcDNATM3.3-TOPO TA Cloning Kit using EcoRI(NEB, R0101S) and XhoI(NEB,R0146S), to construct recombinant vectors for expressing a humanizedantibody.

The constructed vectors were amplified using a Qiagen Maxiprep kit (CatNo. 12662), and the vectors including the heavy chain and the vectorincluding the light chain were added to 293T cells (2.5×10⁷) at a ratioof about 4:1 (about 80 μg:20 μg) with 360 μl of 2 M CaCl₂ and weretransfected. Next, the mixture was cultured in a DMEM medium with 10%(w/v) FBS at 37° C. in 5% (v/v) CO₂ conditions for 5 hours, and thencultured in a DMEM medium without FBS at 37° C. in 5% (v/v) CO₂conditions for 48 hours.

The cultured cells were centrifuged, and 100 ml of each supernatant waspurified using AKTA Prime (GE healthcare). Protein A column (GEhealthcare, 17-0405-03) was placed in the AKTA Prime, and the culturedsolution was flowed at a flow rate of 5 ml/min and was eluted with IgGelution buffer (Thermo Scientific, 21004). The buffer was replaced witha PBS buffer, and thus final humanized antibodies 10D6-HU1, 10D6-HU2,10D6-HU3, and 10D6-HU5 were purified.

Example 7: Incorporation of the Selected CDRs into Humanized Antibodyand Transformation to IgG

The selected CDRs were incorporated into the heavy chain and the lightchain of the humanized antibodies. Polynucleotides encoding the heavychain of the antibodies were synthesized by Bioneer, Inc. so as toconsist of ‘EcoRI-signal sequence-VH-NheI—CH-XhoI’ (SEQ ID NOs: 64-68).Polynucleotides encoding the light chain of the antibodies weresynthesized by Bioneer, Inc. so as to consist of ‘EcoRI-signalsequence-VL-BsiWI-CL-XhoI’ (SEQ ID NOs: 69-71). The polynucleotides (SEQID NOs: 64-68) encoding the heavy chain were respectively cloned into avector of pOptiVEC™-TOPO TA Cloning Kit included in OptiCHO™ AntibodyExpress Kit (Cat no. 12762-019; Invitrogen), and the polynucleotides(SEQ ID NOs: 69-71) encoding the light chain were respectively clonedinto a vector of pcDNA™3.3-TOPOTA Cloning Kit(Cat no. 8300-01), usingEcoRI(NEB, R0101S) and XhoI(NEB, R0146S), to establish vectors forexpressing affinity matured antibodies.

The constructed vectors were amplified using a Qiagen Maxiprep kit (CatNo. 12662), and the vectors including the heavy chain and the vectorincluding the light chain were added to 293T cells (2.5×10⁷) at a ratioof about 4:1 (about 80 μg:20 μg) with 360 μl of 2 M CaCl₂ and weretransfected. Next, the mixture was cultured in a DMEM medium with 10%(w/v) FBS at 37° C. in 5% (v/v) CO₂ conditions for 5 hours, and thencultured in a DMEM medium without FBS at 37° C. in 5% (v/v) CO₂conditions for 48 hours.

The cultured cells were centrifuged, and 100 ml of each supernatant waspurified using AKTA Prime (GE healthcare). Protein A column (GEhealthcare, 17-0405-03) was placed in the AKTA Prime, and the culturedsolution was flowed at a flow rate of 5 ml/min and was eluted with IgGelution buffer (Thermo Scientific, 21004). The buffer was replaced witha PBS buffer, and thus final affinity-matured antibodies h10D6-Opti-1,h10D6-Opti-2, h10D6-Opti-3, and h10D6-Opti-4 were purified.

TABLE 11 Clone Antibody sequence (VH) Antibody sequence (VL)h10D6-OPTI-1 >HU2-6.6 >HU1 QVQLQESGPGLVKPSETLSLTCAVSGYSDIQMTQSPSSLSASVGDRVTITCKASQSV ITSDYAWNWIRQPPGKGLEWMGKISYSSNDVAWYQQKPGKAPKLLIYYASNRYP GKTDYNPSLKSRSTISRDTSKNQFSLKLGVPSRFSGSGSGTDFTLTISSLQPEDFATY SSVTAADTAVYYCARGNFEGAMDYWYCQQDYSSPWTFGQGTKLEIK (SEQ ID GQGTLVTVSS (SEQ ID NO: 52) NO: 57)(coding nucleotide sequence) (coding nucleotide sequence)CAGGTGCAACTGCAGGAGTCAGGCCC GACATCCAGATGACCCAGTCTCCATCCCGGCCTGGTAAAACCTTCTGAAACGCT TCCCTGTCTGCATCTGTAGGAGACAGACTCACTTACCTGTGCCGTTAGTGGATA GTCACCATCACTTGCAAGGCCAGTCAGCTCTATCACTTCCGACTACGCTTGGAA AGTGTGAGTAATGATGTAGCTTGGTATTTGGATTCGGCAGCCTCCAGGCAAAG CAGCAGAAACCAGGGAAAGCCCCTAAGGCTGGAATGGATGGGAAAGATTTCC GCTCCTGATCTATTATGCATCCAATCGCTATTCCGGTAAGACTGACTACAATCCC TACCCTGGGGTCCCATCAAGGTTCAGTAGTCTGAAGAGCAGGTCAACAATCTC GGCAGTGGATCTGGGACAGATTTCACTCAGAGACACCAGCAAGAATCAGTTTT CTCACCATCAGCAGTCTGCAACCTGAACCCTGAAATTGTCCTCGGTGACAGCAG GATTTTGCAACTTACTACTGTCAGCAGCGGATACCGCAGTGTATTATTGCGCCC GATTATAGCTCTCCGTGGACGTTCGGTGCGGTAACTTCGAGGGAGCTATGGAT GGAGGCACCAAGGTGGAAATCAAA (SEQTACTGGGGGCAGGGTACTCTCGTCACT ID NO: 69) GTGAGCAGC (SEQ ID NO: 64)h10D6-OPTI-2 >HU2-6.7 >HU1 QVQLQESGPGLVKPSETLSLTCAVSGYSDIQMTQSPSSLSASVGDRVTITCKASQSV ITSDYAWNWIRQPPGKGLEWMGKINYSNDVAWYQQKPGKAPKLLIYYASNRYP AGNTDYNPSLKSRSTISRDTSKNQFSLKGVPSRFSGSGSGTDFTLTISSLQPEDFATY LSSVTAADTAVYYCARGNFEGAMDYWYCQQDYSSPWTFGQGTKLEIK (SEQ ID GQGTLVTVSS (SEQ ID NO: 53) NO: 57)(coding nucleotide sequence) (coding nucleotide sequence)CAGGTGCAACTGCAGGAGTCAGGCCC GACATCCAGATGACCCAGTCTCCATCCCGGCCTGGTAAAACCTTCTGAAACGCT TCCCTGTCTGCATCTGTAGGAGACAGACTCACTTACCTGTGCCGTTAGTGGATA GTCACCATCACTTGCAAGGCCAGTCAGCTCTATCACTTCCGACTACGCTTGGAA AGTGTGAGTAATGATGTAGCTTGGTATTTGGATTCGGCAGCCTCCAGGCAAAG CAGCAGAAACCAGGGAAAGCCCCTAA GGCTGGAATGGGCTCCTGATCTATTATGCATCCAATCGC ATGGGAAAGATTAACTATGCCGGTAATACCCTGGGGTCCCATCAAGGTTCAGT CACTGACTACAATCCCAGTCTGAAGAGGCAGTGGATCTGGGACAGATTTCACT GCAGGTCAACAATCTCCAGAGACACCCTCACCATCAGCAGTCTGCAACCTGAA AGCAAGAATCAGTTTTCCCTGAAATTGGATTTTGCAACTTACTACTGTCAGCAG TCCTCGGTGACAGCAGCGGATACCGCGATTATAGCTCTCCGTGGACGTTCGGT AGTGTATTATTGCGCCCGCGGTAACTTGGAGGCACCAAGGTGGAAATCAAA (SEQ CGAGGGAGCTATGGATTACTGG ID NO: 69)GGGCAGGGTACTCTCGTCACTGTGAG CAGC (SEQ ID NO: 65)h10D6-OPTI-43 >HU2-6.6 >HU1-6.11 QVQLQESGPGLVKPSETLSLTCAVSGYSDIQMTQSPSSLSASVGDRVTITCKASQSV ITSDYAWNWIRQPPGKGLEWMGKISYSSNDVHWYQQKPGKAPKLLIYYASNRYP GKTDYNPSLKSRSTISRDTSKNQFSLKLGVPSRFSGSGSGTDFTLTISSLQPEDFATY SSVTAADTAVYYCARGNFEGAMDYWYCQQDYSSPWTFGQGTKLEIK (SEQ ID GQGTLVTVSS (SEQ ID NO: 52) NO: 58)(coding nucleotide sequence) (coding nucleotide sequence)CAGGTGCAACTGCAGGAGTCAGGCCC GACATCCAGATGACCCAGTCTCCATCCCGGCCTGGTAAAACCTTCTGAAACGCT TCCCTGTCTGCATCTGTAGGAGACAGACTCACTTACCTGTGCCGTTAGTGGATA GTCACCATCACTTGCAAGGCCAGTCAGCTCTATCACTTCCGACTACGCTTGGAA AGTGTGAGTAATGATGTACATTGGTATTTGGATTCGGCAGCCTCCAGGCAAAG CAGCAGAAACCAGGGAAAGCCCCTAA GGCTGGAATGGGCTCCTGATCTATTATGCATCCAATCGC ATGGGAAAGATTTCCTATTCCGGTAAGTACCCTGGGGTCCCATCAAGGTTCAGT ACTGACTACAATCCCAGTCTGAAGAGGGCAGTGGATCTGGGACAGATTTCACT CAGGTCAACAATCTCCAGAGACACCACTCACCATCAGCAGTCTGCAACCTGAA GCAAGAATCAGTTTTCCCTGAAATTGTGATTTTGCAACTTACTACTGTCAGCAG CCTCGGTGACAGCAGCGGATACCGCAGATTATAGCTCTCCGTGGACGTTCGGT GTGTATTATTGCGCCCGCGGTAACTTCGGAGGCACCAAGGTGGAAATCAAA (SEQ GAGGGAGCTATGGATTACTGG ID NO: 70)GGGCAGGGTACTCTCGTCACTGTGAG CAGC (SEQ ID NO: 64) h10D6-OPTI-55 >HU2-6.7HU1-6.11 QVQLQESGPGLVKPSETLSLTCAVSGYS DIQMTQSPSSLSASVGDRVTITCKASQSVITSDYAWNWIRQPPGKGLEWMGKINY SNDVHWYQQKPGKAPKLLIYYASNRYPAGNTDYNPSLKSRSTISRDTSKNQFSLK GVPSRFSGSGSGTDFTLTISSLQPEDFATYLSSVTAADTAVYYCARGNFEGAMDYW YCQQDYSSPWTFGQGTKLEIK (SEQ IDGQGTLVTVSS (SEQ ID NO: 53) NO: 58) (coding nucleotide sequence)(coding nucleotide sequence) CAGGTGCAACTGCAGGAGTCAGGCCCGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTAAAACCTTCTGAAACGCTTCCCTGTCTGCATCTGTAGGAGACAGA CTCACTTACCTGTGCCGTTAGTGGATAGTCACCATCACTTGCAAGGCCAGTCAG CTCTATCACTTCCGACTACGCTTGGAAAGTGTGAGTAATGATGTACATTGGTAT TTGGATTCGGCAGCCTCCAGGCAAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGCTGGAATGG GCTCCTGATCTATTATGCATCCAATCGCATGGGAAAGATTAACTATGCCGGTAA TACCCTGGGGTCCCATCAAGGTTCAGTCACTGACTACAATCCCAGTCTGAAGA GGCAGTGGATCTGGGACAGATTTCACTGCAGGTCAACAATCTCCAGAGACACC CTCACCATCAGCAGTCTGCAACCTGAAAGCAAGAATCAGTTTTCCCTGAAATTG GATTTTGCAACTTACTACTGTCAGCAGTCCTCGGTGACAGCAGCGGATACCGC GATTATAGCTCTCCGTGGACGTTCGGTAGTGTATTATTGCGCCCGCGGTAACTT GGAGGCACCAAGGTGGAAATCAAA (SEQCGAGGGAGCTATGGATTACTGG ID NO: 70) GGGCAGGGTACTCTCGTCACTGTGAGCAGC (SEQ ID NO: 65) h10D6-OPTI-3 >HU3-6.6 >HU1EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQSVSITSDYAWNWVRQAPGKGLEWMGKIS SNDVAWYQQKPGKAPKLLIYYASNRYPYSGKTDYNPSLKSRSTISRDTSKNTFYL GVPSRFSGSGSGTDFTLTISSLQPEDFATYQMNSLRAEDTAVYYCARGNFEGAMD YCQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 54) NO: 57) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAGCTGGTCGAAAGCGGTGGGACATCCAGATGACCCAGTCTCCATCC GGGACTCGTGCAGCCAGGCGGTTCTCTTCCCTGTCTGCATCTGTAGGAGACAGA TAGATTATCATGTGCCGCATCCGGGTAGTCACCATCACTTGCAAGGCCAGTCAG CTCCATCACCTCTGATTATGCATGGAAAGTGTGAGTAATGATGTAGCTTGGTAT CTGGGTCAGACAAGCCCCCGGAAAGGCAGCAGAAACCAGGGAAAGCCCCTAA GCCTGGAGTGGATGGGGAAGATCTCCGCTCCTGATCTATTATGCATCCAATCGC TATTCAGGGAAGACAGATTATAATCCTTACCCTGGGGTCCCATCAAGGTTCAGT TCGCTGAAAAGCAGATCAACAATTAGGGCAGTGGATCTGGGACAGATTTCACT TAGAGACACTTCTAAAAATACTTTTTACTCACCATCAGCAGTCTGCAACCTGAA CCTCCAGATGAACAGTCTGCGCGCCGGATTTTGCAACTTACTACTGTCAGCAG AAGACACCGCCGTGTACTACTGCGCTGATTATAGCTCTCCGTGGACGTTCGGT AGGGGAAATTTCGAGGGAGCTATGGAGGAGGCACCAAGGTGGAAATCAAA (SEQ CTATTGGGGCCAGGGCACGTTGGTAA ID NO: 69)CCGTGAGCAGC (SEQ ID NO: 66) h10D6-OPTI-4 >HU3-6.7 >HU1EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQSVSITSDYAWNWVRQAPGKGLEWMGKIN SNDVAWYQQKPGKAPKLLIYYASNRYPYAGNTDYNPSLKSRSTISRDTSKNTFYL GVPSRFSGSGSGTDFTLTISSLQPEDFATYQMNSLRAEDTAVYYCARGNFEGAMD YCQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 55) NO: 57) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAACTGGTAGAGTCCGGGGGGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTCCAGCCAGGAGGAAGCCTCCCTGTCTGCATCTGTAGGAGACAGA TGCGGCTCTCTTGTGCCGCCAGCGGGTGTCACCATCACTTGCAAGGCCAGTCAG ATAGTATCACTTCAGATTATGCCTGGAAGTGTGAGTAATGATGTAGCTTGGTAT ATTGGGTCCGCCAGGCCCCCGGGAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGCTTAGAGTGGATGGGTAAAATTAAGCTCCTGATCTATTATGCATCCAATCGC TTACGCAGGCAACACCGACTATAATCTACCCTGGGGTCCCATCAAGGTTCAGT CTTCACTGAAATCTAGATCCACCATCTGGCAGTGGATCTGGGACAGATTTCACT CTAGAGATACAAGTAAGAACACCTTTCTCACCATCAGCAGTCTGCAACCTGAA TACTTGCAGATGAATAGCCTCAGGGCTGATTTTGCAACTTACTACTGTCAGCAG GAAGACACTGCTGTGTACTACTGCGCGATTATAGCTCTCCGTGGACGTTCGGT AAGAGGAAACTTCGAAGGAGCGATGGGGAGGCACCAAGGTGGAAATCAAA (SEQ ATTATTGGGGCCAGGGTACGCTTGTGA ID NO: 69)CAGTGTCCTCT (SEQ ID NO: 67) h10D6-OPTI-16 >HU3-6.6 >HU1-6.11EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQSVSITSDYAWNWVRQAPGKGLEWMGKIS SNDVHWYQQKPGKAPKLLIYYASNRYPYSGKTDYNPSLKSRSTISRDTSKNTFYL GVPSRFSGSGSGTDFTLTISSLQPEDFATYQMNSLRAEDTAVYYCARGNFEGAMD YCQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 54) NO: 58) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAGCTGGTCGAAAGCGGTGGGACATCCAGATGACCCAGTCTCCATCC GGGACTCGTGCAGCCAGGCGGTTCTCTTCCCTGTCTGCATCTGTAGGAGACAGA TAGATTATCATGTGCCGCATCCGGGTAGTCACCATCACTTGCAAGGCCAGTCAG CTCCATCACCTCTGATTATGCATGGAAAGTGTGAGTAATGATGTACATTGGTAT CTGGGTCAGACAAGCCCCCGGAAAGGCAGCAGAAACCAGGGAAAGCCCCTAA GCCTGGAGTGGATGGGGAAGATCTCCGCTCCTGATCTATTATGCATCCAATCGC TATTCAGGGAAGACAGATTATAATCCTTACCCTGGGGTCCCATCAAGGTTCAGT TCGCTGAAAAGCAGATCAACAATTAGGGCAGTGGATCTGGGACAGATTTCACT TAGAGACACTTCTAAAAATACTTTTTACTCACCATCAGCAGTCTGCAACCTGAA CCTCCAGATGAACAGTCTGCGCGCCGGATTTTGCAACTTACTACTGTCAGCAG AAGACACCGCCGTGTACTACTGCGCTGATTATAGCTCTCCGTGGACGTTCGGT AGGGGAAATTTCGAGGGAGCTATGGAGGAGGCACCAAGGTGGAAATCAAA (SEQ CTATTGGGGCCAGGGCACGTTGGTAA ID NO: 70)CCGTGAGCAGC (SEQ ID NO: 66) h10D6-OPTI-17 >HU3-6.7 >HU1-6.11EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQSVSITSDYAWNWVRQAPGKGLEWMGKIN SNDVHWYQQKPGKAPKLLIYYASNRYPYAGNTDYNPSLKSRSTISRDTSKNTFYL GVPSRFSGSGSGTDFTLTISSLQPEDFATYQMNSLRAEDTAVYYCARGNFEGAMD YCQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 55) NO: 58) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAACTGGTAGAGTCCGGGGGGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTCCAGCCAGGAGGAAGCCTCCCTGTCTGCATCTGTAGGAGACAGA TGCGGCTCTCTTGTGCCGCCAGCGGGTGTCACCATCACTTGCAAGGCCAGTCAG ATAGTATCACTTCAGATTATGCCTGGAAGTGTGAGTAATGATGTACATTGGTAT ATTGGGTCCGCCAGGCCCCCGGGAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGCTTAGAGTGGATGGGTAAAATTAAGCTCCTGATCTATTATGCATCCAATCGC TTACGCAGGCAACACCGACTATAATCTACCCTGGGGTCCCATCAAGGTTCAGT CTTCACTGAAATCTAGATCCACCATCTGGCAGTGGATCTGGGACAGATTTCACT CTAGAGATACAAGTAAGAACACCTTTCTCACCATCAGCAGTCTGCAACCTGAA TACTTGCAGATGAATAGCCTCAGGGCTGATTTTGCAACTTACTACTGTCAGCAG GAAGACACTGCTGTGTACTACTGCGCGATTATAGCTCTCCGTGGACGTTCGGT AAGAGGAAACTTCGAAGGAGCGATGGGGAGGCACCAAGGTGGAAATCAAA (SEQ ATTATTGGGGCCAGGGTACGCTTGTGA ID NO: 70)CAGTGTCCTCT (SEQ ID NO: 67) h10D6-OPTI-42 >HU5 >HU1-22QVQLQESGPGLVKPSETLSLTCAVSGYS DIQMTQSPSSLSASVGDRVTITCKASQSVITSDYAWNWIRQPPGKGLEWIGYINYS SNDVHWYQQKPGKAPKLLIYYASNRYPGNTDYNPSLKSRVTISVDTSKNQFSLKL GVPSRFSGSGSGTDFTLTISSLQPEDFATYSSVTAADTAVYYCARGNFEGAMDYW YCQHDYSSPFTFGQGTKLEIK (SEQ IDGQGTLVTVSS (SEQ ID NO: 56) NO: 59) (coding nucleotide sequence)(coding nucleotide sequence) CAGGTGCAGCTGCAGGAGTCGGGCCCGACATCCAGATGACCCAGTCTCCATCC AGGACTGGTGAAGCCTTCGGAGACCCTCCCTGTCTGCATCTGTAGGAGACAGA TGTCCCTCACCTGCGCTGTCTCTGGTTGTCACCATCACTTGCAAGGCCAGTCAG ACTCCATCACCAGTGATTATGCCTGGAAGTGTGAGTAATGATGTACATTGGTAT ACTGGATCCGGCAGCCCCCAGGGAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGGCTGGAGTGGATTGGGTACATAAAGCTCCTGATCTATTATGCATCCAATCGC CTACAGTGGTAACACTGACTACAACCTACCCTGGGGTCCCATCAAGGTTCAGT CATCTCTCAAAAGTCGAGTCACCATATGGCAGTGGATCTGGGACAGATTTCACT CAGTAGACACGTCCAAGAACCAGTTCCTCACCATCAGCAGTCTGCAACCTGAA TCCCTGAAGCTGAGCTCTGTGACCGCCGATTTTGCAACTTACTACTGTCAGCAT GCAGACACGGCCGTGTATTACTGTGCGATTATAGCTCTCCGTTCACGTTCGGTG GAGAGGTAACTTCGAAGGTGCTATGGGAGGCACCAAGGTGGAAATCAAA (SEQ ACTACTGGGGTCAAGGAACGCTTGTG ID NO: 71)ACAGTGTCCTCT (SEQ ID NO: 68) (In Table 11, the bold letters are CDR1,CDR2, and CDR3 in sequence)

Example 8: Analysis of Binding Affinity of Selected Antibodies

The binding affinity (KD values) of the antibodies to human Ang2 proteinwas measured by an SPR method using a BIAcore T100 (GE Healthcare). 25μg/ml anti-His antibody was immobilized on a CM5 sensor chip (GEhealthcare) using a pH 5.0 acetate solution and an amine coupling kit(GE Healthcare). 6 μg/ml of a recombinant hAng2 (C-His, R&D Systems)protein was flowed onto the chip to be captured at 100 to 200 RU levels.The antibodies obtained in the above examples were diluted serially totwice each time starting from 100 nM concentration and it was eachflowed onto the chip to allow it to be bound to (on), dissociated from(off), and regenerated (using 10 mM NaOH solution) from the antigencaptured on the sensor chip, thereby to measure antigen-antibodyaffinity. The KD values were calculated from the values of k_(on)k_(off), and the results are as shown in the following Table 12.

TABLE 12 Antibody kon (1/Ms) koff (1/s) KD (nM) m10D6 2.410 × 10⁴ 1.932× 10⁻⁴ 8 10D6-HU1 3.082 × 10⁴ 0.002599 84 10D6-HU2 7.298 × 10⁴ 0.00346447 10D6-HU3 4.503 × 10⁴ 0.001938 43 10D6-HU5 4.856 × 10⁴ 0.003115 64h10D6-OPTI-1 4.737 × 10⁵ 3.209 × 10⁻⁴ 0.68 h10D6-OPTI-2 4.237 × 10⁵1.488 × 10⁻⁴ 0.34 h10D6-OPTI-43 1.531 × 10⁶ 5.760 × 10⁻⁴ 0.38h10D6-OPTI-55 6.210 × 10⁵ 8.489 × 10⁻⁵ 0.14 h10D6-OPTI-3 6.239 × 10⁵3.070 × 10⁻⁴ 0.49 h10D6-OPTI-4 7.357 × 10⁵ 2.460 × 10⁻⁴ 0.33h10D6-OPTI-16 4.794 × 10⁵ 4.434 × 10⁻⁴ 0.92 h10D6-OPTI-17 4.600 × 10⁵3.503 × 10⁻⁴ 0.76 h10D6-OPTI-42 3.358 × 10⁵ 2.862 × 10⁻⁴ 0.85

As shown in Table 12, the affinity to Ang2 of the mouse antibody 10D6 isabout 8 nM, the affinities to Ang2 of the 5 affinity-matured andhumanized antibodies are from about 0.14 nM to about 0.92 nM. Theresults indicate that the affinity to Ang2 can be improved at leastabout 5 times up to about 37 times in the affinity-matured antibodies inan IgG form transformed from a scFv form.

Example 9: Analysis of In Vitro Biological Property of the SelectedAffinity-Matured Antibodies-Akt Phosphorylation

To examine whether the humanized and/or affinity-matured 10D6 antibodiescan induce activation of downstream signaling as well as Tie2 receptor,the levels of Akt phosphorylation in HUVEC (ATCC) cells treated withAng2 and each of the antibodies (see Table 11) of Example 7 weremeasured and compared to that of the case treated with Ang2 only. HUVEC(ATCC) cells (2×10⁴ cells) were cultured in 96 well plate using EGM-2medium (Lonza) at 37° C., and when they reached 80˜90% confluency, themedia were replaced with serum-free medium (Lonza) and cultured at 37°C. for 6 hours. The cultured cells were treated with a mixture preparedby mixing 6 nM or 1.2 nM of each of the anti-Ang2 antibodies of Example7 with 4 nM of Ang2 protein (R&D systems) and letting them stand for 20min. and further cultured for 30 min.

The phosphorylation of Akt which participates in downstream signaling ofTie2 receptor was examined using PATHSCAN® Phospho-Akt ChemiluminescentSandwich ELISA Kit (Cell signaling, #7134). The cells were washed usingPBS, treated with 30 μl of a lysis buffer (Roche), to be subjected tocell lysis at 4° C. for 30 minutes. Then, 30 μl of diluent buffer (Cellsignaling) was added to each well and sufficiently mixed with pipet, and50 μl of the diluted product was collected and transferred to aphosphor-Akt Ab coated microwell, to react at room temperature for 2hours. After 2 hours, the well was washed with 1× washing buffer (Cellsignaling) four times, and treated with 50 μl of Akt1 detection antibodysolution (Cell signaling), to react at room temperature for one hour. Asthe same process, the well was washed, and reacted with 50 μl ofHRP-conjugated secondary antibody (Cell signaling) at room temperaturefor 30 minutes. As the same process, the well was washed, and treatedwith 50 μl of a mixture solution of luminol/enhancer solution(GEhealthcare) and stable peroxide buffer(GE healthcare) at the ratio of1:1 (v/v). Then the plate was placed in a luminometer (Envision 2104plate reader, Perkin Elmer), to measure a relative light unit (RLU).

The obtained results are shown in FIG. 2. As seen in FIG. 2, thehumanized and/or affinity-matured antibodies of Example 7 induce thedownstream signaling more intensively compared to mouse antibody 10D6.

Example 10: Synthesis of a Polynucleotide for Preparing an scFv ofHumanized Antibody of 10D6(Opti-1)

The gene for preparing scFv of a humanized 10D6 antibody was designedusing the heavy chain variable region and the light chain variableregion of humanized 10D6 antibody Opti-1. The heavy chain variableregion (amino acid sequence: Hu2 6.6 (SEQ ID NO: 52); coding nucleotidesequence: SEQ ID NO: 64), and the light chain variable region (aminoacid sequence: SEQ ID NO: Hu1(SEQ ID NO: 57); coding nucleotidesequence: SEQ ID NO: SEQ ID NO: 69) were linked to form a ‘VH-linker-VL’construct, and the linker was designed so as to have the amino acidsequence of ‘GGGGSGGGGSGGGGS(SEQ ID NO: 76)’. The polynucleotide (SEQ IDNO: 83) encoding the designed scFv (‘VH-linker-VL’; SEQ ID NO: 82) ofantibody 10D6 opti-1 was synthesized by Bioneer, Inc.

Example 11: Preparation of Gene Library for the Secondary AffinityMaturation

11.1. Selection of Target CDR and Preparation of Primers

To perform affinity maturation of antibody 10D6 opti-1, threecomplementary determining regions (CDRs) were defined from the preparedantibody 10D6 opti-1 according to the ‘Kabat numbering’ rule. The CDRsare summarized in Table 13:

TABLE 13 CDR amino acid sequence CDR-L1 KASQSVSNDVA (SEQ ID NO: 4)CDR-L2 YASNRYP (SEQ ID NO: 5) CDR-L3 QQDYSSPWT (SEQ ID NO: 6)

For use in the introduction of random sequences into the CDRs of theantibody, primers were designed as follows. Conventionally, N codonswere utilized to introduce bases at the same ratio (25% A, 25% G, 25% C,25% T) into desired sites of mutation. In this experiment, theintroduction of random bases into the CDRs of 10D6 was conducted in sucha manner that, of the three nucleotides per codon in the wild-typepolynucleotide encoding each CDR, the first and second nucleotidesconserved over 85% of the entire sequence while the other threenucleotides were introduced at the same percentage (each 5%) and thatthe same possibility was imparted to the third nucleotide (33% G, 33% C,33% T).

11.2. Construction of Gene Library of scFv of 10D6 Opti-1 Antibody

The construction of antibody gene libraries through the introduction ofrandom sequences was carried out using the primers synthesized in thesame manner as in Example 11.1. Two PCR products were obtained using apolynucleotide covering the 10D6 opti-1 scFv(SEQ ID NO: 83) as atemplate (see FIG. 4), and were subjected to overlap extension PCR togive scFv library genes for 10D6 antibodies in which only desired CDRswere mutated. 10⁷˜10⁸ libraries targeting each of the six CDRs preparedfrom the scFv library genes were constructed.

The affinity for Ang2 of each library was compared to that of thewild-type. Most libraries were lower in affinity for Ang2, compared tothe wild-type. However, in some mutants, the affinity for Ang2 wasretained.

Example 12: Selection of Antibody with Improved Affinity from Libraries

Among the scFv libraries provided in Example 11, the scFv fragmentsshowing upper 1.0 percent of affinity to Ang-2 were selected, and thisprocess was repeated four times. The nucleotide sequence of each of theselected scFv was analyzed. The obtained nucleotide sequences aresummarized in Table 14, and were converted into IgG forms (a heavy chainconstant region: constant region of human IgG1, a light chain constantregion: constant region of human KAPPA Chain). Four antibodies whichwere respectively produced from clones 10D6_VL-Hu1-2.1, 10D6_VL-Hu1-2.4,10D6_VL-Hu1-2.7, 10D6_VL-Hu1-2.8 were used in the subsequentexperiments.

TABLE 14 Library Clones constructed CDR sequence 10D6_VL-Hu1-2.1 CDR-L1KASQFVSTDVH (SEQ ID NO: 17) 10D6_VL-Hu1-2.4 CDR-L2 YASIPYP(SEQ ID NO: 18) 10D6_VL-Hu1-2.7 CDR-L1 + L2 KASQSVSNDVH (SEQ IDNO: 16) + YASIPYP (SEQ ID NO: 18) 10D6_VL-Hu1-2.8 CDR-L1 + L2KASQFVSTDVH (SEQ ID NO: XX) + YASIPYP (SEQ ID NO: 18)

Example 13: Incorporation of the Selected CDRs into Humanized Antibodyand Transformation to IgG

The selected CDRs were incorporated into the heavy chain and the lightchain of the humanized antibodies. The heavy chain was derived from theantibody cloned with Hu2-6.6 or Hu3-6.6. Polynucleotides encoding thelight chain of the antibodies were synthesized by Bioneer, Inc. so as toconsist of ‘EcoRI-signal sequence-VL-BsiWI-CL-XhoI’ (see Table 15). Thepolynucleotides encoding the heavy chain were respectively cloned into avector of pOptiVEC™-TOPO TA Cloning Kit included in OptiCHO™ AntibodyExpress Kit (Cat no. 12762-019; Invitrogen), and the polynucleotidesencoding the light chain were respectively cloned into a vector ofpcDNA™3.3-TOPOTA Cloning Kit(Cat no. 8300-01), using EcoRI(NEB, R0101S)and XhoI(NEB, R0146S), to establish vectors for expressing affinitymatured antibodies.

The constructed vectors were amplified using a Qiagen Maxiprep kit (CatNo. 12662), and the vectors including the heavy chain and the vectorincluding the light chain were added to 293T cells (2.5×10⁷) at a ratioof about 4:1 (about 80 μg:20 μg) with 360 μl of 2 M CaCl₂ and weretransfected. Next, the mixture was cultured in a DMEM medium with 10%(w/v) FBS at 37° C. in 5% (v/v) CO₂ conditions for 5 hours, and thencultured in a DMEM medium without FBS at 37° C. in 5% (v/v) CO₂conditions for 48 hours.

The cultured cells were centrifuged, and 100 ml of each supernatant waspurified using AKTA Prime (GE healthcare). Protein A column (GEhealthcare, 17-0405-03) was placed in the AKTA Prime, and the culturedsolution was flowed at a flow rate of 5 ml/min and was eluted with IgGelution buffer (Thermo Scientific, 21004). The buffer was replaced witha PBS buffer, and thus final affinity-matured antibodies (hereinafter,named as h10D6-Opti-63, h10D6-Opti-64, h10D6-Opti-65, h10D6-Opti-66,h10D6-Opti-67, h10D6-Opti-71, h10D6-Opti-68, h10D6-Opti-70,h10D6-Opti-72, and h10D6-Opti-73) were purified.

TABLE 15 Clone Antibody sequence (VH) Antibody sequence (VL)h10D6-OPTI-63 >HU2-6.6 >10D6_VL-Hu1-2.1 QVQLQESGPGLVKPSETLSLTCAVSGYSDIQMTQSPSSLSASVGDRVTITCKASQFV ITSDYAWNWIRQPPGKGLEWMGKISYSSTDVHWYQQKPGKAPKLLIYYASNRYP GKTDYNPSLKSRSTISRDTSKNQFSLKLGVPSRFSGSGSGTDFTLTISSLQPEDFATY SSVTAADTAVYYCARGNFEGAMDYWYCQQDYSSPWTFGQGTKLEIK (SEQ ID GQGTLVTVSS (SEQ ID NO: 52) NO: 60)(coding nucleotide sequence) (coding nucleotide sequence)CAGGTGCAACTGCAGGAGTCAGGCCC GACATCCAGATGACCCAGTCTCCATCCCGGCCTGGTAAAACCTTCTGAAACGC TCCCTGTCTGCATCTGTAGGAGACAGATCTCACTTACCTGTGCCGTTAGTGGAT GTCACCATCACTTGCAAGGCCAGTCAGACTCTATCACTTCCGACTACGCTTGGA TTCGTGAGTACTGATGTACATTGGTATATTGGATTCGGCAGCCTCCAGGCAAA CAGCAGAAACCAGGGAAAGCCCCTAA GGGCTGGAATGGGCTCCTGATCTATTATGCATCCAATCGC ATGGGAAAGATTTCCTATTCCGGTAATACCCTGGGGTCCCATCAAGGTTCAGT GACTGACTACAATCCCAGTCTGAAGAGGCAGTGGATCTGGGACAGATTTCACT GCAGGTCAACAATCTCCAGAGACACCCTCACCATCAGCAGTCTGCAACCTGAA AGCAAGAATCAGTTTTCCCTGAAATTGATTTTGCAACTTACTACTGTCAGCAG GTCCTCGGTGACAGCAGCGGATACCGGATTATAGCTCTCCGTGGACGTTCGGT CAGTGTATTATTGCGCCCGCGGTAACGGAGGCACCAAGGTGGAAATCAAA (SEQ TTCGAGGGAGCTATGGATTACTGG ID NO: 72)GGGCAGGGTACTCTCGTCACTGTGAG CAGC (SEQ ID NO: 64) h10D6-OPTI-64 >HU2-6.6DIQMTQSPSSLSASVGDRVTITCKASQSV QVQLQESGPGLVKPSETLSLTCAVSGYSSNDVAWYQQKPGKAPKLLIYYASNRYP ITSDYAWNWIRQPPGKGLEWMGKISYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY GKTDYNPSLKSRSTISRDTSKNQFSLKLYCGQDYASPWTFGQGTKLEIK (SEQ ID SSVTAADTAVYYCARGNFEGAMDYW NO: 87)GQGTLVTVSS (SEQ ID NO: 52) (coding nucleotide sequence)(coding nucleotide sequence) CAGGTGCAACTGCAGGAGTCAGGCCCGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTAAAACCTTCTGAAACGCTCCCTGTCTGCATCTGTAGGAGACAGA TCTCACTTACCTGTGCCGTTAGTGGATGTCACCATCACTTGCAAGGCCAGTCAG ACTCTATCACTTCCGACTACGCTTGGAAGTGTGAGTAATGATGTAGCT ATTGGATTCGGCAGCCTCCAGGCAAATGGTATCAGCAGAAACCAGGGAAAGC GGGCTGGAATGG CCCTAAGCTCCTGATCTATTATGCATCCATGGGAAAGATTTCCTATTCCGGTAA AACCGATACCCTGGGGTCCCATCAAGGGACTGACTACAATCCCAGTCTGAAGA TTCAGTGGCAGTGGATCTGGGACAGATGCAGGTCAACAATCTCCAGAGACACC TTCACTCTCACCATCAGCAGTCTGCAAAGCAAGAATCAGTTTTCCCTGAAATT CCTGAAGATTTTGCAACTTACTACTGTGTCCTCGGTGACAGCAGCGGATACCG GGACAGGATTATGCCTCTCCGTGGACGCAGTGTATTATTGCGCCCGCGGTAAC TTCGGTGGAGGCACCAAGGTGGAAATCTTCGAGGGAGCTATGGATTACTGG AAA (SEQ ID NO: 88) GGGCAGGGTACTCTCGTCACTGTGAGCAGC (SEQ ID NO: 64) h10D6-OPTI-65 >HU2-6.6 >10D6_VL-Hu1-2.4QVQLQESGPGLVKPSETLSLTCAVSGYS DIQMTQSPSSLSASVGDRVTITCKASQSVITSDYAWNWIRQPPGKGLEWMGKISYS SNDVAWYQQKPGKAPKLLIYYASIPYPGGKTDYNPSLKSRSTISRDTSKNQFSLKL VPSRFSGSGSGTDFTLTISSLQPEDFATYYSSVTAADTAVYYCARGNFEGAMDYW CQQDYSSPWTFGQGTKLEIK (SEQ IDGQGTLVTVSS (SEQ ID NO: 52) NO: 61) (coding nucleotide sequence)(coding nucleotide sequence) CAGGTGCAACTGCAGGAGTCAGGCCCGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTAAAACCTTCTGAAACGCTCCCTGTCTGCATCTGTAGGAGACAGA TCTCACTTACCTGTGCCGTTAGTGGATGTCACCATCACTTGCAAGGCCAGTCAG ACTCTATCACTTCCGACTACGCTTGGAAGTGTGAGTAATGATGTAGCTTGGTAT ATTGGATTCGGCAGCCTCCAGGCAAACAGCAGAAACCAGGGAAAGCCCCTAA GGGCTGGAATGG GCTCCTGATCTATTATGCATCCATCCCAATGGGAAAGATTTCCTATTCCGGTAA TACCCTGGGGTCCCATCAAGGTTCAGTGACTGACTACAATCCCAGTCTGAAGA GGCAGTGGATCTGGGACAGATTTCACTGCAGGTCAACAATCTCCAGAGACACC CTCACCATCAGCAGTCTGCAACCTGAAAGCAAGAATCAGTTTTCCCTGAAATT GATTTTGCAACTTACTACTGTCAGCAGGTCCTCGGTGACAGCAGCGGATACCG GATTATAGCTCTCCGTGGACGTTCGGTCAGTGTATTATTGCGCCCGCGGTAAC GGAGGCACCAAGGTGGAAATCAAA (SEQTTCGAGGGAGCTATGGATTACTGG ID NO: 73) GGGCAGGGTACTCTCGTCACTGTGAGCAGC (SEQ ID NO: 64) h10D6-OPTI-66 >HU2-6.6DIQMTQSPSSLSASVGDRVTITCKASQSV QVQLQESGPGLVKPSETLSLTCAVSGYSSNDVHWYQQKPGKAPKLLIYYASNRYP ITSDYAWNWIRQPPGKGLEWMGKISYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY GKTDYNPSLKSRSTISRDTSKNQFSLKLYCGQDYSAPWTFGQGTKLEIK (SEQ ID SSVTAADTAVYYCARGNFEGAMDYW NO: 89)GQGTLVTVSS (SEQ ID NO: 52) (coding nucleotide sequence)(coding nucleotide sequence) CAGGTGCAACTGCAGGAGTCAGGCCCGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTAAAACCTTCTGAAACGCTCCCTGTCTGCATCTGTAGGAGACAGA TCTCACTTACCTGTGCCGTTAGTGGATGTCACCATCACTTGCAAGGCCAGTCAG ACTCTATCACTTCCGACTACGCTTGGAAGTGTGAGTAATGATGTACAC ATTGGATTCGGCAGCCTCCAGGCAAATGGTATCAGCAGAAACCAGGGAAAGC GGGCTGGAATGG CCCTAAGCTCCTGATCTATTATGCATCCATGGGAAAGATTTCCTATTCCGGTAA AACCGATACCCTGGGGTCCCATCAAGGGACTGACTACAATCCCAGTCTGAAGA TTCAGTGGCAGTGGATCTGGGACAGATGCAGGTCAACAATCTCCAGAGACACC TTCACTCTCACCATCAGCAGTCTGCAAAGCAAGAATCAGTTTTCCCTGAAATT CCTGAAGATTTTGCAACTTACTACTGTGTCCTCGGTGACAGCAGCGGATACCG GGACAGGATTATTCTGCCCCGTGGACGCAGTGTATTATTGCGCCCGCGGTAAC TTCGGTGGAGGCACCAAGGTGGAAATCTTCGAGGGAGCTATGGATTACTGG AAA (SEQ ID NO: 90) GGGCAGGGTACTCTCGTCACTGTGAGCAGC (SEQ ID NO: 64) h10D6-OPTI-67 >HU2-6.6 >10D6_VL-Hu1-2.7QVQLQESGPGLVKPSETLSLTCAVSGYS DIQMTQSPSSLSASVGDRVTITCKASQSVITSDYAWNWIRQPPGKGLEWMGKISYS SNDVHWYQQKPGKAPKLLIYYASIPYPGGKTDYNPSLKSRSTISRDTSKNQFSLKL VPSRFSGSGSGTDFTLTISSLQPEDFATYYSSVTAADTAVYYCARGNFEGAMDYW CQQDYSSPWTFGQGTKLEIK (SEQ IDGQGTLVTVSS (SEQ ID NO: 52) NO: 62) (coding nucleotide sequence)(coding nucleotide sequence) CAGGTGCAACTGCAGGAGTCAGGCCCGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTAAAACCTTCTGAAACGCTCCCTGTCTGCATCTGTAGGAGACAGA TCTCACTTACCTGTGCCGTTAGTGGATGTCACCATCACTTGCAAGGCCAGTCAG ACTCTATCACTTCCGACTACGCTTGGAAGTGTGAGTAATGATGTACATTGGTAT ATTGGATTCGGCAGCCTCCAGGCAAACAGCAGAAACCAGGGAAAGCCCCTAA GGGCTGGAATGG GCTCCTGATCTATTATGCATCCATCCCAATGGGAAAGATTTCCTATTCCGGTAA TACCCTGGGGTCCCATCAAGGTTCAGTGACTGACTACAATCCCAGTCTGAAGA GGCAGTGGATCTGGGACAGATTTCACTGCAGGTCAACAATCTCCAGAGACACC CTCACCATCAGCAGTCTGCAACCTGAAAGCAAGAATCAGTTTTCCCTGAAATT GATTTTGCAACTTACTACTGTCAGCAGGTCCTCGGTGACAGCAGCGGATACCG GATTATAGCTCTCCGTGGACGTTCGGTCAGTGTATTATTGCGCCCGCGGTAAC GGAGGCACCAAGGTGGAAATCAAA (SEQTTCGAGGGAGCTATGGATTACTGG ID NO: 74) GGGCAGGGTACTCTCGTCACTGTGAGCAGC (SEQ ID NO: 64) h10D6-OPTI-71 >HU2-6.6 10D6_VL-Hu1-2.8QVQLQESGPGLVKPSETLSLTCAVSGYS DIQMTQSPSSLSASVGDRVTITCKASQFVITSDYAWNWIRQPPGKGLEWMGKISYS STDVHWYQQKPGKAPKLLIYYASIPYPGGKTDYNPSLKSRSTISRDTSKNQFSLKL VPSRFSGSGSGTDFTLTISSLQPEDFATYYSSVTAADTAVYYCARGNFEGAMDYW CQQDYSSPWTFGQGTKLEIK (SEQ IDGQGTLVTVSS (SEQ ID NO: 52) NO: 63) (coding nucleotide sequence)(coding nucleotide sequence) CAGGTGCAACTGCAGGAGTCAGGCCCGACATCCAGATGACCCAGTCTCCATCC CGGCCTGGTAAAACCTTCTGAAACGCTCCCTGTCTGCATCTGTAGGAGACAGA TCTCACTTACCTGTGCCGTTAGTGGATGTCACCATCACTTGCAAGGCCAGTCAG ACTCTATCACTTCCGACTACGCTTGGATTCGTGAGTACTGATGTACATTGGTAT ATTGGATTCGGCAGCCTCCAGGCAAACAGCAGAAACCAGGGAAAGCCCCTAA GGGCTGGAATGG GCTCCTGATCTATTATGCATCCATCCCAATGGGAAAGATTTCCTATTCCGGTAA TACCCTGGGGTCCCATCAAGGTTCAGTGACTGACTACAATCCCAGTCTGAAGA GGCAGTGGATCTGGGACAGATTTCACTGCAGGTCAACAATCTCCAGAGACACC CTCACCATCAGCAGTCTGCAACCTGAAAGCAAGAATCAGTTTTCCCTGAAATT GATTTTGCAACTTACTACTGTCAGCAGGTCCTCGGTGACAGCAGCGGATACCG GATTATAGCTCTCCGTGGACGTTCGGTCAGTGTATTATTGCGCCCGCGGTAAC GGAGGCACCAAGGTGGAAATCAAA (SEQTTCGAGGGAGCTATGGATTACTGG ID NO: 75) GGGCAGGGTACTCTCGTCACTGTGAGCAGC (SEQ ID NO: 64) h10D6-OPTI-68 >HU3-6.6 >10D6_VL-Hu1-2.1EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQFVSITSDYAWNWVRQAPGKGLEWMGKIS STDVHWYQQKPGKAPKLLIYYASNRYPYSGKTDYNPSLKSRSTISRDTSKNTFYL GVPSRFSGSGSGTDFTLTISSLQPEDFATYQMNSLRAEDTAVYYCARGNFEGAMD YCQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 54) NO: 60) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAGCTGGTCGAAAGCGGTGGGACATCCAGATGACCCAGTCTCCATCC GGGACTCGTGCAGCCAGGCGGTTCTCTCCCTGTCTGCATCTGTAGGAGACAGA TTAGATTATCATGTGCCGCATCCGGGTGTCACCATCACTTGCAAGGCCAGTCAG ACTCCATCACCTCTGATTATGCATGGATTCGTGAGTACTGATGTACATTGGTAT ACTGGGTCAGACAAGCCCCCGGAAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGCCTGGAGTGGATGGGGAAGATCTCGCTCCTGATCTATTATGCATCCAATCGC CTATTCAGGGAAGACAGATTATAATCTACCCTGGGGTCCCATCAAGGTTCAGT CTTCGCTGAAAAGCAGATCAACAATTGGCAGTGGATCTGGGACAGATTTCACT AGTAGAGACACTTCTAAAAATACTTTCTCACCATCAGCAGTCTGCAACCTGAA TTACCTCCAGATGAACAGTCTGCGCGGATTTTGCAACTTACTACTGTCAGCAG CCGAAGACACCGCCGTGTACTACTGCGATTATAGCTCTCCGTGGACGTTCGGT GCTAGGGGAAATTTCGAGGGAGCTATGGAGGCACCAAGGTGGAAATCAAA (SEQ GGACTATTGGGGCCAGGGCACGTTGG ID NO: 72)TAACCGTGAGCAGC (SEQ ID NO: 66) h10D6-OPTI-70 >HU3-6.6 >10D6_VL-Hu1-2.4EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQSVSITSDYAWNWVRQAPGKGLEWMGKIS SNDVAWYQQKPGKAPKLLIYYASIPYPGYSGKTDYNPSLKSRSTISRDTSKNTFYL VPSRFSGSGSGTDFTLTISSLQPEDFATYYQMNSLRAEDTAVYYCARGNFEGAMD CQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 54) NO: 61) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAGCTGGTCGAAAGCGGTGGGACATCCAGATGACCCAGTCTCCATCC GGGACTCGTGCAGCCAGGCGGTTCTCTCCCTGTCTGCATCTGTAGGAGACAGA TTAGATTATCATGTGCCGCATCCGGGTGTCACCATCACTTGCAAGGCCAGTCAG ACTCCATCACCTCTGATTATGCATGGAAGTGTGAGTAATGATGTAGCTTGGTAT ACTGGGTCAGACAAGCCCCCGGAAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGCCTGGAGTGGATGGGGAAGATCTCGCTCCTGATCTATTATGCATCCATCCCA CTATTCAGGGAAGACAGATTATAATCTACCCTGGGGTCCCATCAAGGTTCAGT CTTCGCTGAAAAGCAGATCAACAATTGGCAGTGGATCTGGGACAGATTTCACT AGTAGAGACACTTCTAAAAATACTTTCTCACCATCAGCAGTCTGCAACCTGAA TTACCTCCAGATGAACAGTCTGCGCGGATTTTGCAACTTACTACTGTCAGCAG CCGAAGACACCGCCGTGTACTACTGCGATTATAGCTCTCCGTGGACGTTCGGT GCTAGGGGAAATTTCGAGGGAGCTATGGAGGCACCAAGGTGGAAATCAAA (SEQ GGACTATTGGGGCCAGGGCACGTTGG ID NO: 73)TAACCGTGAGCAGC (SEQ ID NO: 66) h10D6-OPTI-72 >HU3-6.6 >10D6_VL-Hu1-2.7EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQSVSITSDYAWNWVRQAPGKGLEWMGKIS SNDVHWYQQKPGKAPKLLIYYASIPYPGYSGKTDYNPSLKSRSTISRDTSKNTFYL VPSRFSGSGSGTDFTLTISSLQPEDFATYYQMNSLRAEDTAVYYCARGNFEGAMD CQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 54) NO: 62) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAGCTGGTCGAAAGCGGTGGGACATCCAGATGACCCAGTCTCCATCC GGGACTCGTGCAGCCAGGCGGTTCTCTCCCTGTCTGCATCTGTAGGAGACAGA TTAGATTATCATGTGCCGCATCCGGGTGTCACCATCACTTGCAAGGCCAGTCAG ACTCCATCACCTCTGATTATGCATGGAAGTGTGAGTAATGATGTACATTGGTAT ACTGGGTCAGACAAGCCCCCGGAAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGCCTGGAGTGGATGGGGAAGATCTCGCTCCTGATCTATTATGCATCCATCCCA CTATTCAGGGAAGACAGATTATAATCTACCCTGGGGTCCCATCAAGGTTCAGT CTTCGCTGAAAAGCAGATCAACAATTGGCAGTGGATCTGGGACAGATTTCACT AGTAGAGACACTTCTAAAAATACTTTCTCACCATCAGCAGTCTGCAACCTGAA TTACCTCCAGATGAACAGTCTGCGCGGATTTTGCAACTTACTACTGTCAGCAG CCGAAGACACCGCCGTGTACTACTGCGATTATAGCTCTCCGTGGACGTTCGGT GCTAGGGGAAATTTCGAGGGAGCTATGGAGGCACCAAGGTGGAAATCAAA (SEQ GGACTATTGGGGCCAGGGCACGTTGG ID NO: 74)TAACCGTGAGCAGC (SEQ ID NO: 66) h10D6-OPTI-73 >HU3-6.6 10D6_VL-Hu1-2.8EVQLVESGGGLVQPGGSLRLSCAASGY DIQMTQSPSSLSASVGDRVTITCKASQFVSITSDYAWNWVRQAPGKGLEWMGKIS STDVHWYQQKPGKAPKLLIYYASIPYPGYSGKTDYNPSLKSRSTISRDTSKNTFYL VPSRFSGSGSGTDFTLTISSLQPEDFATYYQMNSLRAEDTAVYYCARGNFEGAMD CQQDYSSPWTFGQGTKLEIK (SEQ IDYWGQGTLVTVSS (SEQ ID NO: 54) NO: 63) (coding nucleotide sequence)(coding nucleotide sequence) GAGGTTCAGCTGGTCGAAAGCGGTGGGACATCCAGATGACCCAGTCTCCATCC GGGACTCGTGCAGCCAGGCGGTTCTCTCCCTGTCTGCATCTGTAGGAGACAGA TTAGATTATCATGTGCCGCATCCGGGTGTCACCATCACTTGCAAGGCCAGTCAG ACTCCATCACCTCTGATTATGCATGGATTCGTGAGTACTGATGTACATTGGTAT ACTGGGTCAGACAAGCCCCCGGAAAGCAGCAGAAACCAGGGAAAGCCCCTAA GGCCTGGAGTGGATGGGGAAGATCTCGCTCCTGATCTATTATGCATCCATCCCA CTATTCAGGGAAGACAGATTATAATCTACCCTGGGGTCCCATCAAGGTTCAGT CTTCGCTGAAAAGCAGATCAACAATTGGCAGTGGATCTGGGACAGATTTCACT AGTAGAGACACTTCTAAAAATACTTTCTCACCATCAGCAGTCTGCAACCTGAA TTACCTCCAGATGAACAGTCTGCGCGGATTTTGCAACTTACTACTGTCAGCAG CCGAAGACACCGCCGTGTACTACTGCGATTATAGCTCTCCGTGGACGTTCGGT GCTAGGGGAAATTTCGAGGGAGCTATGGAGGCACCAAGGTGGAAATCAAA (SEQ GGACTATTGGGGCCAGGGCACGTTGG ID NO: 75)TAACCGTGAGCAGC (SEQ ID NO: 66) (In Table 15, the bold letters are CDR1,CDR2 and CDR3 in sequence)

Example 14: Analysis of Binding Affinity of Selected Antibodies

The binding affinity (KD values) of the antibodies to human Ang2 proteinwas measured by an SPR method using a BIAcore T100 (GE Healthcare). 25μg/ml anti-His antibody was immobilized on a CM5 sensor chip (GEhealthcare) using a pH 5.0 acetate solution and an amine coupling kit(GE Healthcare). 6 μg/ml of a recombinant hAng2 (C-His, R&D Systems)protein was flowed onto the chip to be captured at 100 to 200 RU levels.The antibodies obtained in the above examples were diluted serially totwice each time starting from 100 nM concentration and it was eachflowed onto the chip to allow it to be bound to (on), dissociated from(off), and regenerated (using 10 mM NaOH solution) from the antigencaptured on the sensor chip, thereby to measure antigen-antibodyaffinity. The KD values were calculated from the values of k_(on)k_(off), and the results are as shown in the following Table 16.

TABLE 16 Antibody kon (1/Ms) koff (1/s) KD (M) h10D6-OPTI-63 2.676 × 10⁶7.421 × 10⁻⁵ 2.773 × 10⁻¹¹ h10D6-OPTI-65 4.960 × 16⁵ 2.250 × 10⁻⁵ 4.536× 10⁻¹² h10D6-OPTI-67 2.080 × 16⁶ 2.684 × 10⁻⁷ 1.291 × 10⁻¹³h10D6-OPTI-68 5.355 × 10⁵ 1.696 × 10⁻⁴ 3.168 × 10⁻¹⁰ h10D6-OPTI-70 2.650× 10⁵ 1.159 × 10⁻⁴ 4.374 × 10⁻¹⁰

As shown in Table 16, all the affinity-matured and humanized antibodiesshow high affinity to Ang2 from about 0.000129 nM to about 0.43 nM.

Example 15: Analysis of In Vitro Biological Property of the SelectedAffinity-Matured Antibodies-Akt Phosphorylation

To examine whether the humanized and/or affinity-matured 10D6 antibodiescan induce activation of downstream signaling as well as Tie2 receptor,the levels of Akt phosphorylation in HUVEC (ATCC) cells treated withAng2 and each of the antibodies (see Table 15) of Example 13 weremeasured and compared to that of the case treated with Ang2 only. HUVEC(ATCC) cells (2×10⁴ cells) were cultured in 96 well plate using EGM-2medium (Lonza) at 37° C., and when they reached 80˜90% confluency, themedia were replaced with serum-free medium (Lonza) and cultured at 37°C. for 6 hours. The cultured cells were treated with a mixture preparedby mixing 6 nM, 1.2 nM, or 0.24 nM of each of the anti-Ang2 antibodiesof Example 13 with 4 nM of Ang2 protein (R&D systems) and letting themstand for 20 min. and further cultured for 30 min.

The phosphorylation of Akt which participates in downstream signaling ofTie2 receptor was examined using PATHSCAN® Phospho-Akt ChemiluminescentSandwich ELISA Kit (Cell signaling, #7134). The cells were washed usingPBS, treated with 30 μl of a lysis buffer (Roche), to be subjected tocell lysis at 4° C. for 30 minutes. Then, 30 μl of diluent buffer (Cellsignaling) was added to each well and sufficiently mixed with pipet, and50 μl of the diluted product was collected and transferred to aphosphor-Akt Ab coated microwell, to react at room temperature for 2hours. After 2 hours, the well was washed with 1× washing buffer (Cellsignaling) four times, and treated with 50 μl of Akt1 detection antibodysolution (Cell signaling), to react at room temperature for one hour. Asthe same process, the well was washed, and reacted with 50 ul ofHRP-conjugated secondary antibody (Cell signaling) at room temperaturefor 30 minutes. As the same process, the well was washed, and treatedwith 50 ul of a mixture solution of luminol/enhancer solution(GEhealthcare) and stable peroxide buffer(GE healthcare) at the ratio of1:1 (v/v). Then the plate was placed in a luminometer (Envision 2104plate reader, Perkin Elmer), to measure a relative light unit (RLU).

The obtained results are shown in FIG. 5. As seen in FIG. 5, thehumanized and/or affinity-matured antibodies pf Example 13 induce thedownstream signaling more intensively compared to mouse antibody 10D6.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of activating Tie2 in a patientsuffering from a disease related to Ang2 overexpression, angiogenesis,or an increase in vascular permeability in a subject, the methodcomprising administering an anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need thereof, wherein the anti-Ang2antibody or an antigen-binding fragment thereof binds to Ang2 and bindsto Tie2 receptor via Ang2, and comprises: a heavy chain variable regioncomprising a polypeptide (CDR-H1) comprising SEQ ID NO: 1, a polypeptide(CDR-H2) comprising SEQ ID NO: 20, and a polypeptide (CDR-H3) comprisingSEQ ID NO: 3; and a light chain variable region comprising a polypeptide(CDR-L1) comprising SEQ ID NO: 21, a polypeptide (CDR-L2) comprising SEQID NO: 22, and a polypeptide (CDR-L3) comprising SEQ ID NO: 23; or: aheavy chain variable region comprising a polypeptide (CDR-H1) comprisingSEQ ID NO: 1, a polypeptide (CDR-H2) comprising SEQ ID NO: 14, and apolypeptide (CDR-H3) comprising SEQ ID NO: 3; and a light chain variableregion comprising a polypeptide (CDR-L1) comprising SEQ ID NO: 4, apolypeptide (CDR-L2) comprising SEQ ID NO: 5, and a polypeptide (CDR-L3)comprising SEQ ID NO: 91; or: a heavy chain variable region comprising apolypeptide (CDR-H1) comprising SEQ ID NO: 1, a polypeptide (CDR-H2)comprising SEQ ID NO: 14, and a polypeptide (CDR-H3) comprising SEQ IDNO: 3; and a light chain variable region comprising a polypeptide(CDR-L1) comprising SEQ ID NO: 16, a polypeptide (CDR-L2) comprising SEQID NO: 5, and a polypeptide (CDR-L3) comprising SEQ ID NO: 92; with theproviso that the anti-Ang2 antibody or an antigen-binding fragmentthereof does not comprise all of a polypeptide (CDR-H1) comprising SEQID NO: 1, a polypeptide (CDR-H2) comprising SEQ ID NO: 2, a polypeptide(CDR-H3) comprising SEQ ID NO: 3, a polypeptide (CDR-L1) comprising SEQID NO: 4, a polypeptide (CDR-L2) comprising SEQ ID NO: 5, and apolypeptide (CDR-L3) comprising SEQ ID NO: 6; and wherein SEQ ID NO: 20,21, 22 and 23 are as follows: X₁IX₃YX₅GX₇TDYNPSLKS (SEQ ID NO: 20),wherein: X₁ is Y or K; X₃ is N or S; X₅ is S or A; and X₇ is N or K;KASQX₅VSX₈DVX₁₁ (SEQ ID NO: 21), wherein: X₅ is S or F; X₈ is N or T;and X₁₁ is A or H; YASX₄X₅YP (SEQ ID NO: 22), wherein: X₄ is N or I; andX₅ is R or P; QX₂DYSSPX₈T (SEQ ID NO: 23); wherein: X₂ is Q or H; and X₈is W or F.
 2. The method of claim 1, wherein the disease related to Ang2overexpression, angiogenesis, or an increase in vascular permeability iscancer, cancer metastasis, ocular blood vessel disorder, inflammatorydisorder, infectious disorder, cardiovascular disorder, renal disease,sepsis, asthma, edema, or hereditary hemorrhagic telangiectasia (HHT).3. The method of claim 1, further comprising administering Ang2 to thesubject.
 4. The method of claim 1, wherein the disease is myocardialinfarction, angina, cerebral infarction, stroke, Buerger's disease,avascular necrosis, foot ulcer, or erectile dysfunction.
 5. The methodof claim 4, further comprising administering Ang2 to the subject.
 6. Themethod of claim 1, wherein the anti-Ang2 antibody or antigen-bindingfragment thereof comprises: a heavy chain variable region comprising apolypeptide (CDR-H1) comprising SEQ ID NO: 1, a polypeptide (CDR-H2)comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 14, and 15, and a polypeptide (CDR-H3) comprising SEQ IDNO: 3; and a light chain variable region comprising a polypeptide(CDR-L1) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 16, and 17, a polypeptide (CDR-L2)comprising SEQ ID NO: 5 or 18, and a polypeptide (CDR-L3) comprising SEQID NO: 6 or 19; with the proviso that the anti-Ang2 antibody or anantigen-binding fragment thereof does not comprise all of a polypeptide(CDR-H1) comprising SEQ ID NO: 1, a polypeptide (CDR-H2) comprising SEQID NO: 2, a polypeptide (CDR-H3) comprising SEQ ID NO: 3, a polypeptide(CDR-L1) comprising SEQ ID NO: 4, a polypeptide (CDR-L2) comprising SEQID NO: 5, and a polypeptide (CDR-L3) comprising SEQ ID NO: 6 at the sametime.
 7. The method of claim 1, wherein the anti-Ang2 antibody orantigen-binding fragment thereof comprises: a heavy chain variableregion comprising one of SEQ ID NOs: 52 to 56, and a light chainvariable region comprising one of SEQ ID NOs: 57 to
 63. 8. The method ofclaim 1, wherein the anti-Ang2 antibody or antigen-binding fragmentthereof comprises: a heavy chain variable region comprising SEQ ID NO:52, and a light chain variable region comprising SEQ ID NO: 87 or
 89. 9.The method of claim 1, wherein the anti-Ang2 antibody or antigen bindingfragment is an scFv, (scFv)2, scFv-Fc, Fab, Fab′ or F(ab′)2 antigenbinding antibody fragment.
 10. The method of claim 1, wherein theanti-Ang2 antibody is a humanized antibody, an affinity-maturedantibody, or a combination thereof.
 11. The method of claim 4, whereinthe anti-Ang2 antibody or antigen-binding fragment thereof comprises: aheavy chain variable region comprising a polypeptide (CDR-H1) comprisingSEQ ID NO: 1, a polypeptide (CDR-H2) comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 14, and 15, and apolypeptide (CDR-H3) comprising SEQ ID NO: 3; and a light chain variableregion comprising a polypeptide (CDR-L1) comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 16, and17, a polypeptide (CDR-L2) comprising SEQ ID NO: 5 or 18, and apolypeptide (CDR-L3) comprising SEQ ID NO: 6 or 19; with the provisothat the anti-Ang2 antibody or an antigen-binding fragment thereof doesnot comprise all of a polypeptide (CDR-H1) comprising SEQ ID NO: 1, apolypeptide (CDR-H2) comprising SEQ ID NO: 2, a polypeptide (CDR-H3)comprising SEQ ID NO: 3, a polypeptide (CDR-L1) comprising SEQ ID NO: 4,a polypeptide (CDR-L2) comprising SEQ ID NO: 5, and a polypeptide(CDR-L3) comprising SEQ ID NO: 6 at the same time.
 12. The method ofclaim 4, wherein the anti-Ang2 antibody or antigen-binding fragmentthereof comprises: a heavy chain variable region comprising one of SEQID NOs: 52 to 56, and a light chain variable region comprising one ofSEQ ID NOs: 57 to
 63. 13. The method of claim 4, wherein the anti-Ang2antibody or antigen-binding fragment thereof comprises: a heavy chainvariable region comprising SEQ ID NO: 52, and a light chain variableregion comprising SEQ ID NO: 87 or
 89. 14. The method of claim 4,wherein the anti-Ang2 antibody or antigen binding fragment is an scFv,(scFv)2, scFv-Fc, Fab, Fab′ or F(ab′)2 antigen binding antibodyfragment.
 15. The method of claim 4, wherein the anti-Ang2 antibody is ahumanized antibody, an affinity-matured antibody, or a combinationthereof.